Small and intermediate conductance, calcium-activated potassium channels and uses thereof

ABSTRACT

This invention relates to small and intermediate conductance, calcium-activated potassium channel proteins. More specifically the invention relates to compositions and methods for making and detecting calcium-activated potassium channel proteins and the nucleic acids encoding calcium-activated potassium channel proteins. The invention also provides methods and compositons for assaying compounds which increase or decrease potassium ion flux through a calcium-activated potassium channel.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/254,590, filed May 24, 1999, now U.S. Pat. No. 6,797,486, which is a35 USC § 371 US national phase of PCT application PCT/US97/16033, filedSep. 10, 1997, the disclosure of which is herein incorporated byreference in its entirety. This application claims the benefit of thefiling date of U.S. Ser. No. 60/026,451, filed on Sep. 11, 1996; U.S.Ser. No. 60/040,052, filed on Mar. 7, 1997; and U.S. Ser. No 60/045,233,filed on Apr. 17, 1997.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with United States Government support underGrant No. 1R01NS31872-01A1, awarded by the National Institutes ofHealth. The United States Government has certain rights in thisinvention.

FIELD OF THE INVENTION

The present invention relates to compositions relating to, and methodsfor identifying, small conductance (SK) and intermediate conductance(IK), calcium-activated potassium channels. The invention furtherprovides a method to assay for compounds that increase or decreasepotassium ion flux through calcium-activated channels.

BACKGROUND OF THE INVENTION

Calcium-activated potassium currents are found in a wide variety ofanimal cells such as nervous, muscular, glandular or epithelial tissueand from the immune system. The channels regulating these currents openand allow the escape of potassium as the internal calcium concentrationincreases. This outward flow of potassium ions makes the interior of thecell more negative, counteracting depolarizing voltages applied to thecell.

Two distinct classes of calcium-activated K⁺ channels (K_(ca) channels)have been described. Large conductance calcium-activated K⁺ channels (BKchannels) are gated by the concerted actions of internal calcium ionsand membrane potential, and have a unit conductance between 100 and 220pS. Small (SK) and intermediate (IK) conductance calcium-activated K⁺channels are gated solely by internal calcium ions, with a unitconductance of 2-20 and 20-85 pS, respectively, and are more sensitiveto calcium than are BK channels (for review see Latorre et al., 1989,Ann Rev Phys, 51, 385-399.). In addition, each type of K_(ca) channelshows a distinct pharmacological profile. All three classes are widelyexpressed, and their activity hyperpolarizes the membrane potential.Members of the BK (Atkinson et al., 1991, Science, 253, 551-555.;Adelman et al., 1992 Neuron, 9, 209-216.; Butler, 1993, Science, 261,221-224) and SK (Kohler et al., 1996, Science, 273, 1709-1714.)subfamilies have been cloned and expressed in heterologous cell typeswhere they recapitulate the fundamental properties of their nativecounterparts.

In vertebrate neurons action potentials are followed by anafterhyperpolarization (AHP) that may persist for several seconds andhave profound consequences for the firing pattern of the neuron.Alterations in the AHP have been implicated in seizure activity (Algeret al., J. Physiol. 399:191-205 (1988)) and learning and memory (deJonge et al., Exp. Br. Res. 80:456-462 (1990)). The AHP is composed oftwo prominent components, a fast component (fAHP) which mediates spikefrequency at the onset of a burst, and a subsequent slow component(sAHP) which is responsible for spike-frequency adaptation (Nicoll,Science 241:545-551 (1988)).

Each component of the AHP is kinetically distinct and is due toactivation of different calcium-activated potassium channels. Activationof large-conductance (100-200 picoSiemens (pS)), voltage- andcalcium-activated potassium channels (BK channels) underlies the fAHP(Lancaster et al, J. Physiol. 389:187-203 (1987); Viana et al., J.Neurophysiol. 69:2150-2163 (1993)) which develops rapidly (1-2 ms) anddecays within tens of milliseconds. The channels underlying the sAHP aresmall conductance, calcium activated, potassium channels (SK channels)which differ from BK channels, being more calcium-sensitive, are notvoltage-gated, and possessing a smaller unit conductance (Lancaster etal., J. Neurosci. 11:23-30 (1991); Sah. J. Neurophysiol. 74:1772-1776(1995)).

The fAHP and the sAHP also differ in their pharmacology. The fAHP isblocked by low concentrations of external tetraethylammonium (TEA) andcharybdotoxin (CTX), in accord with the pharmacology of the BK channels.Lancaster et al, J. Physiol. 389:187-203 (1987); Viana et al., J.Neurophysiol. 69:2150-2163 (1993); Butler et al., Science 261:221-224(1993). In contrast, the sAHP is insensitive to CTX, but fall into twoclasses regarding sensitivity to the bee venom peptide toxin, apamin.For example, in hippocampal pyramidal neurons, the sAHP is insensitiveto apamin (Lancaster et al., J. Neurophysiol. 55:1268-1282 (1986)),while in hippocampal interneurons and vagal neurons it is blocked bynanomolar concentrations of the toxin (Sah, J. Neurophysiol.74:1772-1776 (1995); Zhang et al., J. Physiol. 488:661-672 (1995)).

In addition to its role in neuronal cells, non-voltage gated,apamin-sensitive potassium channels activated by submicromolarconcentrations of calcium have also been described from peripheral celltypes, including skeletal muscle (Blatz et al., Nature 323:718-720(1986)), gland cells (Tse et al., Science 255:462-464 (1992); Park, J.Physiol. 481:555-570 (1994)) and T-lymphocytes (Grissmer et al., J. Gen.Physiol. 99:63-84 (1992)).

For example, SK channels have been suggested to represent the apaminreceptor found in muscle membrane of patients with myotonic musculardystrophy. Renaud et al., Nature 319:678-680 (1986)). Also, Grissmer etal. (J. Gen. Physiol. 99:63-84 (1992)) report that CTX insensitive,apamin sensitive calcium-activated potassium channels were identified ina human leukemic T cell line and suggest that calcium-activatedpotassium channels play a supporting role during T-cell activation bysustaining dynamic patterns of calcium signaling. And in many cells, SKchannels are activated as a result of neurotransmitter or hormoneaction. Haylett et al., in Potassium Channels: Structure,Classification, Function and Therapeutic Potential (Cook, N. S., ed.),pp. 71-95, John Wiley and Sons, 1990). Intermediate channels play a rolein the physiology of red blood cells.

Intermediate conductance, calcium activated potassium channels have beenpreviously described in the literature by their electrophysiology. TheGardos channel is opened by submicromolar concentrations of internalcalcium and has a rectifying unit conductance, ranging from 50 pS at−120 mV to 13 pS at 120 mV (symmetrical 120 mM K⁺; Christophersen, 1991,J. Membrane Biol., 119, 75-83.). It is blocked by charybdotoxin (CTX)but not the structurally related peptide iberiotoxin (IBX), both ofwhich block BK channels (Brugnara et al., 1995a, J. Membr. Biol., 147,71-82). Apamin, a potent blocker of certain native (Vincent et al.,1975, J. Biochem., 14, 2521.; Blatz and Magleby, 1986, Nature, 323,718-720.) and cloned SK channels do not block IK channels (de-Allie etal., 1996, Br. J. Pharm., 117,479-487). The Gardos channel is alsoblocked by some imidazole compounds, such as clotrimazole, but notketoconazole (Brugnara et al., 1993, J. Clin. Invest., 92,520-526). Theelectrophysiological and pharmacological properties of the Gardoschannel show that it belongs to the IK subfamily of this invention.

IK channels have been described in a variety of other cell types.Principle cells of the rat cortical collecting duct segregate differentclasses of K⁺ channels to the luminal and basolateral membranes. IKchannels are present in the basolateral membrane where they promote therecirculation of K⁺ across this membrane, elevating the activity of theNa⁺+K⁺-ATPase and thereby Na⁺ reabsorption into the blood (Hirsch andSchlatter, 1995, Pflügers Arch.—Eur. J. Physiol., 449, 338-344.) IKchannels have also been implicated in the microvasculature of the kidneywhere they may be responsible for the vasodilatory effects of bradykinin(Rapacon et al., 1996). In brain capillary endothelial cells, IKchannels are activated by endothelin, produced by neurons and glia,shunting excess K⁺ into the blood (Renterghem et al., 1995, J.Neurochem., 65, 1274-1281). Neutrophil granulocytes, mobile phagocyticcells which defend against microbial invaders, undergo a largedepolarization subsequent to agonist stimulation, and IK channels havebeen implicated in repolarizing the stimulated granulocyte (Varnai etal., 1993, J. Physiol., 472, 373-390.). IK channels have also beenidentified in both resting and activated human T-lymphocytes. Grissmeret al. 1993, J. Gen. Physiol. 102, 601-630 reported that IK channelswere blocked by low nanomolar concentrations of charybdotoxin, showedlittle or no voltage dependence, and were insensitive to apamin. Thischannel has also been identified in human erythrocytes, where it playsan important role in intracellular volume homeostasis (Joiner, C. H.1993, Am. J. Physiol. 264: C251-270 and in smooth muscle (VanRentergnem, C. et al. 1996, J. Neurochemistry 65, 1274-1281.

Thus, it appears that SK and IK channels comprise a subfamily ofcalcium-activated potassium channels which play key physiological rolesin many cell types. Accordingly, given the key role of SK and IKchannels in a wide variety of physiological functions, what is needed inthe art is the identification of novel SK and IK channel proteins andthe nucleic acids encoding them. Additionally, what is needed aremethods of identifying compounds which increase or decrease SK and IKchannel currents for their use in the treatment or regulation of:learning and memory disorders, seizures, myotonic dystrophies, immuneresponses, and neurotransmitter or hormone secretions. The presentinvention provides these and other advantages.

SUMMARY OF THE INVENTION

In a first broad context, this invention provides for novel proteins andtheir corresponding nucleic acids where the proteins are defined asmonomers of calcium activated potassium ion channels. The monomers havea molecular weight of between 40 and 80 kDa and have units ofconductance of between 2 and 80 pS when the monomer is in the polymericform as expressed in Xenopus oocytes. In addition, the monomerspecifically binds to antibodies generated against SEQ ID NO:30 or 42.

In another aspect, the present invention relates to an isolated nucleicacid encoding at least 15 contiguous amino acids of a calcium-activatedpotassium channel protein. The SK channel protein has a sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ IDNO:43, and SEQ ID NO:47 and conservatively modified variants of SEQ IDNOS:1, 2, 3, 4, 19, 20, 32, 43 or 47.

In some embodiments, the isolated nucleic acid encodes acalcium-activated potassium channel protein having a conductance of atleast 2 pS when expressed in a Xenopus oocyte, a molecular weight ofbetween 40 and 100 kilodaltons (kd), and selectively hybridizes, understringent hybridization conditions, with SK or IK encoding nucieic acidsuch as SEQ ID NO:13 in a human genomic library or SEQ ID NO:14 in a ratgenomic library. In other embodiments, the isolated nucleic acidencoding the calcium-activated potassium channel protein encodes aprotein having a sequence selected from the group consisting of: SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47. In preferred embodimentsthe nucleic acid has a sequence selected from the group consisting of:SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:31, SEQ ID NO:44, and SEQ ID NO:48.

In another aspect, the present invention relates to an isolatedcalcium-activated potassium channel protein having at least 15contiguous amino acids of a sequence selected from the group consistingof: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47, andconservatively modified variants of SEQ ID NOS:1, 2, 3, 4, 19, 20, 32,43, or 47, wherein the variant specifically reacts, underimmunologically reactive conditions, with an antibody reactive to aprotein selected from the group consisting of. SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:30, SEQID NO:32, SEQ ID NO:43, and SEQ ID NO:47.

In a broad embodiment, the calcium-activated potassium channel proteinis defined as having a conductance of at least 2 pS and a molecularweight of between 40 and 100 Kd. In other embodiments, thecalcium-activated potassium channel protein has an amino acid sequenceselected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ IDNO:43, and SEQ ID NO:47.

In another aspect, the present invention is directed to an antibodyspecifically reactive, under immunologically reactive conditions, to acalcium-activated potassium channel protein, where the protein has asequence selected from the group consisting of: SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:32, SEQ ID NO:43, and SEQ ID NO:47 in preferred embodiments, theantibody is limited to a monoclonal antibody.

In yet another aspect, the present invention relates to an expressionvector comprising a nucleic acid encoding a monomer of acalcium-activated potassium channel where the monomer has a sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ IDNO:43, and SEQ ID NO:47, and conservatively modified variants of SEQ IDNOS:1, 2, 3, 4, 19, 20, 32, 43, or 47 wherein the modified variant is aprotein having a conductance of at least 2 pS when expressed in aXenopus oocyte, a molecular weight of between 40 and 100 kd, andspecifically reacts, under immunologically reactive conditions, with anantibody reactive to a full-length protein selected from the groupconsisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47.

In another aspect, the present invention relates to a host celltransfected with a vector comprising a nucleic acid encoding a monomerof a calcium-activated potassium channel protein where the protein has asequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, SEQID NO:47, and conservatively modified variants of SEQ ID NO:1, 2, 3, 4,19, 20, 32, 43 or 47 wherein the modified variant is a protein having aconductance of at least 2 pS when expressed in a Xenopus oocyte, amolecular weight of between 40 and 100 Kd, and specifically reacts,under immunologically reactive conditions, with an antibody reactive toa full-length protein selected from the group consisting of: SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:32, SEQ ID NO:43, and SEQ ID NO:47. Typically, the host cell iscultured under conditions permitting expression of the nucleic acidencoding the calcium-activated potassium channel protein.

In yet a further aspect, the present invention relates to an isolatednucleic acid sequence of at least 15 nucleotides in length whichspecifically hybridizes, under stringent conditions, to a nucleic acidencoding a calcium-activated potassium channel protein, where theprotein is selected from the group consisting of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:32, SEQ ID NO:43, and SEQ ID NO:47.

In an additional aspect, the present invention is directed to a methodfor detecting the presence of a calcium-activated potassium channelprotein in a biological sample. The method comprises contacting thebiological sample with an antibody, wherein the antibody specificallyreacts, under immunologically reactive conditions, to ancalcium-activated potassium channel protein having a sequence selectedfrom the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQID NO:47 and allowing the antibody to bind to the protein underimmunologically reactive conditions, wherein detection of the boundantibody indicates the presence of the channel protein.

In yet another aspect, the present invention provides a method fordetecting the presence, in a biological sample, of a nucleic acidsequence encoding a calcium-activated potassium channel protein of atleast 25 amino acids in length. The method comprises contacting thebiological sample, under stringent hybridization conditions, with anucleic acid probe comprising a nucleic acid segment that selectivelyhybridizes to a nucleic acid encoding the channel protein having asequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQID NO:43, and SEQ ID NO:47; allowing the nucleic acid encoding thechannel protein to selectively hybridize to the probe to form ahybridization complex, wherein detection of the hybridization complex isan indication of the presence of the nucleic acid sequence in thesample. In some embodiments, the hybridization conditions are moderatestringency hybridization conditions. In another embodiment, the calciumactivated channel protein is at least 400 amino acid residues in lengthand when expressed in oocytes has a conductance of at least 2 pS. In afurther embodiment, the nucleic acid probes comprises at least 250contiguous nucleotides encoding a subsequence within the small orintermediate calcium-activated potassium channel protein core region.

In a further aspect, the present invention relates to an isolatedcalcium-activated potassium channel encoded by a nucleic acid amplifiedby primers which selectively hybridize, under stringent hybridizationconditions, to the same nucleic acid sequence as primers selected fromthe group consisting of: for hSK1. SEQ ID NO:5 and SEQ ID NO:6; for rSK2SEQ ID NO:7 and SEQ ID NO:8; for endogenous rSK3, SEQ ID NO:9 and SEQ IDNO:10; for rSK1, SEQ ID NO:11 and SEQ ID NO:12; for hSK2, SEQ ID NO:23and SEQ ID NO:24; for hSK3, SEQ ID NO:25 and SEQ ID NO:26; and for hIKthe following primer pairs will amplify a probe that is selective foridentifying hIK1 from a human genomic or cDNA library: 5′GCCGTGCGTGCAGGATTTAGG 3′ (SEQ ID NO:34) and 5′CCAGAGGCCAAGCGTGAGGCC 3′(SEQ ID NO:35) yielding a probe of about 270 bases or 5′TCCAAGATGCACATGATCCTG 3′ (SEQ ID NO:36) and 5′ GGACTGCTGGCTGGGTTCTGG 3′(SEQ ID NO:37) yielding a probe of about 165 bases. For amplification ofa full length hIK1 either of the following two primer pairs will work:5′ ATGGGCGGGGATCTGGTGCTTG 3′ (SEQ ID NO:38) and 5′CTACTTGGACTGCTGGCTGGGTTC 3′ (SEQ ID NO:39) or 5′ ATGGGCGGGGATCTGGTGCTTGG3′ (includes codon of initiator methionine) (SEQ ID NO:40) and 5′GGGTCCAGCTACTTGGACTGCTG 3′ (includes stop codon for end of translation)(SEQ ID NO:41).

In yet another aspect, the present invention relates to a method ofidentifying a compound which increases or decreases the potassium ionflux through a small or intermediate conductance, calcium-activatedpotassium channel, with the provisio that the compound is notclotrimizole. The method comprises the steps of contacting the compoundwith a eukaryotic host cell in which has been expressed a nucleic acidencoding a calcium-activated potassium channel having a sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ IDNO:43, SEQ ID NO:47 and conservatively modified variants thereof,wherein said conservatively modified variant specifically binds toantibodies specifically reactive with an antigen having an amino acidsequence selected from the group consisting of: SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:32, SEQ ID NO:43, and SEQ ID NO:47, have a conductance of at least 2pS, and a molecular weight between 40 and 100 kilodaltons; anddetermining the increased or decreased flux of potassium ions throughsaid channel. In preferred embodiments, the increased or decreased fluxof potassium ions is determined by measuring the electrical current orflux of ions, or indirectly the change in voltage induced by the changein current or flux of ions, across the cell membrane of said eukaryotichost cell. In a particularly preferred embodiment, the channel proteinhas a sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:32, SEQ ID NO:43, and SEQ ID NO:47. In another preferred embodiment,the channel protein is recombinant.

In a further aspect, the present invention relates to an isolatedeukaryotic nucleic acid encoding a calcium-activated potassium channelprotein of at least 400 amino acid residues in length, wherein thecalcium-activated channel protein comprises an amino acid sequencehaving at least 55 to 60% similarity over the length of a core region ofa protein selected from the group consisting of: SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:32, SEQ ID NO:43, and SEQ ID NO:47 and wherein the channel proteinhas a conductance of at least 2 pS. In some embodiments, the presentinvention is directed to the protein encoded by the aforementionedisolated eukaryotic nucleic acid. In other embodiments, the isolatednucleic acid encoding the calcium-activated channel protein has at least85% sequence similarity over a comparison window of 20 contiguous aminoacid residues within the core region.

In a further aspect, the present invention is directed to a vectorcomprising an isolated eukaryotic nucleic acid encoding acalcium-activated potassium channel protein of at least 400 amino acidresidues in length, wherein the channel protein comprises an amino acidsequence having at least 55% similarity over the length of a core regionof a protein selected from the group consisting of: SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:32, SEQ ID NO:43, and SEQ ID NO:47, and wherein the channel proteinhas a conductance of at least 2 pS. Typically, the vector is transfectedinto a host cell which is cultured under conditions permittingexpression of the isolated eukaryotic nucleic acid encoding the channelprotein.

In a further aspect, present invention is directed to a method ofidentifying a compound that increases or decreases the potassium ionflux through a calcium-activated potassium channel. The methodscomprises the steps of contacting the compound with a eukaryotic hostcell in which has been expressed a calcium-activated potassium channelprotein of at least 400 amino acid residues in length, wherein thechannel protein has an amino acid sequence having at least 55%similarity over the length of a core region of a protein selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ IDNO:47, and wherein the channel protein has a conductance of at least 2pS; and determining the increased or decreased flux of potassium ionsthrough the channel protein. In some embodiments the increased ordecreased flux of potassium ions is determined by measuring theelectrical current across the cell membrane of the eukaryotic host cell.

In another aspect, the present invention provides in a computer system amethod of screening for mutations of SK and IK genes, the methodcomprising the steps of: (i) receiving input of a first nucleic acidsequence encoding a calcium-activated channel protein having a sequenceselected from the group consisting of SEQ ID NOS:1, 2, 3, 4, 19, 20, 32,43, 47 and conservatively modified versions thereof; (ii) comparing thefirst nucleic acid sequence with a second nucleic acid sequence havingsubstantial identity to the first nucleic acid sequence; and (iii)identifying nucleotide differences between the first and second nucleicacid sequences. In one embodiment, the second nucleic acid sequence isassociated with a disease state.

In another aspect, the invention provides in a computer system, a methodfor identifying a three-dimensional structure of SK and IK proteins, themethod comprising the steps of: (i) receiving input of an amino acidsequence of a calcium-activated channel protein or a nucleotide sequenceof a gene encoding the protein, the protein having an amino acidsequence selected from the group consisting of SEQ ID NOS:1, 2, 3, 4,19, 20, 32, 43, 47, and conservatively modified versions thereof; and(ii) generating a three-dimensional structure of the protein encoded bythe amino acid sequence. In one embodiment, the amino acid sequence is aprimary structure and the generating step includes the steps of forminga secondary structure from the primary structure using energy termsencoded by the primary structure and forming a tertiary structure fromthe secondary structure using energy terms encoded by said secondarystructure. In another embodiment, the generating step includes the stepof forming a quaternary structure from the tertiary structure usingnianistrophy terms encoded by the tertiary structure. In anotherembodiment, the method further comprises the step of identifying regionsof the three-dimensional structure of the protein that bind to ligandsand using the regions to identify ligands that bind to the protein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel isolated, small conductance,calcium-activated potassium (SK) channels, intermediate conductance,calcium-activated potassium (IK) channels (collectively,“calcium-activated potassium channels”), and isolated nucleic acidsencoding SK and IK channels (i.e., SK and IK channel nucleic acids). Thedistribution, function, and pharmacology define these new classes ofchannels as SK or IK channels.

Expression of isolated SK or IK channel protein encoding nucleic acidsin a host cell provides a composition which can be used to identifycompounds that increase or decrease potassium ion flux through smallconductance, calcium-activated potassium (SK) channels or intermediateconductance, calcium-activated potassium (IK) channels, respectively.Since SK channels underlie the slow component of theafterhyperpolarization (sAHP) of neurons, alteration of neuronal sAHPprovides a means to inhibit epileptic seizures or modulate learning ormemory disorders.

Calcium activated, SK channels are also implicated in T-cell activation.Thus, increasing or decreasing SK channel currents provides a means toinhibit or potentiate the immune response. Moreover, SK channels areassociated with hormone and neurotransmitter secretions. Accordingly,altering SK channel currents provides a means to regulate cellular orglandular secretions and thereby treat imbalances thereof.

Calcium activated intermediate channels (IK) are also believed to playan important physiological role particularly in peripheral tissues. Forexample, intermediate channels are reported in red blood cells, and, inpart, contribute to cell dehydration, a process that is exacerbated insickle cell anemia.

The invention also relates to subsequences of isolated small conductanceand intermediate conductance, calcium-activated potassium channels andfor isolated nucleic acids encoding SK and IK channel proteins. Isolatednucleic acids coding for SK or IK channel proteins provide utility asprobes for identification of aberrant transcription products orincreased or decreased transcription levels of genes coding for SK or IKchannels. Assaying for increased or decreased transcription can be usedin drug screening protocols. Likewise, SK or IK channel proteins can beused as immunogens to generate antibodies for use in immunodiagnosticassays of increased or decreased expression of calcium-activatedpotassium channels in drug screening assays.

Definitions

Units, prefixes, and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges are inclusiveof the numbers defining the range. The terms defined below are morefully defined by reference to the specification as a whole.

The terms “nucleic acid” “probe”, or “primer” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues of natural nucleotides that hybridize to nucleic acids inmanner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence includes the perfectcomplementary sequence thereof. Eukaryotic nucleic acids are nucleicacids from eukaryotic cells, preferably cells of multicellulareukaryotes.

The term “recombinant” when used with reference to a cell, or protein,nucleic acid, or vector, includes reference to a cell, protein, ornucleic acid, or vector, that has been modified by the introduction of aheterologous nucleic acid or the alteration of a native nucleic acid toa form not native to that cell, or that the cell is derived from a cellso modified. Thus, for example, recombinant cells express genes andproteins that are not found within the native (non-recombinant) form ofthe cell or express native genes that are otherwise abnormallyexpressed, under expressed or not expressed at all.

The term “subsequence” in the context of a referenced nucleic acidsequence includes reference to a contiguous sequence from the nucleicacid having fewer nucleotides in length than the referenced nucleicacid. In the context of a referenced protein, polypeptide, or peptidesequence (collectively, “protein”), “subsequence” refers to a contiguoussequence from the referenced protein having fewer amino acids than thereferenced protein.

The terms “identical” or “sequence identity” in the context of twonucieic acid or polypeptide sequences includes reference to the residuesin the two sequences which are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g. chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. Where sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Means for making thisadjustment are well-known to those of skill in the art. Typically thisinvolves scoring a conservative substitution as a partial rather than afull mismatch, thereby increasing the percentage sequence identity.Thus, for example, where an identical amino acid is given a score of 1and a non-conservative substitution is given a score of zero, aconservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., according tothe algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17 (1988) e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif., USA).

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison may be conducted by the local homology algorithm of Smithand Waterman (1981) Adv. Appl. Math. 2: 482; by the homology alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443; by thesearch for similarity method of Pearson and Lipman (1988) Proc. Natl.Acad. Sci. USA 85: 2444; by computerized implementations of thesealgorithms (including, but not limited to CLUSTAL in the PC/Gene programby lntelligenetics, Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup (GCG), 575 Science Dr., Madison, Wis., USA); the CLUSTAL programis well described by Higgins and Sharp (1988) Gene, 73: 237-244 andHiggins and Sharp (1989) CABIOS 5: 151-153; Corpet, et al. (1988)Nucleic Acids Research 16, 10881-90; Huang, et al. (1992) ComputerApplications in the Biosciences 8, 155-65, and Pearson, et al. (1994)Methods in Molecular Biology 24, 307-31. Alignment is also oftenperformed by inspection and manual alignment.

The terms “substantial identity” or “similarity” of polynucleotidesequences means that a polynucleotide comprises a sequence that has atleast 60% sequence identity, preferably at least 80%, more preferably atleast 90% and most preferably at least 95%, compared to a referencesequence using the programs described above (preferably BLAST) usingstandard parameters. One indication that two nucleic acid sequences aresubstantially identical is that the polypeptide which the first nucleicacid encodes is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

Another indication that two nucieic acid sequences have substantiallyidentity is that the two molecules hybridize to each other under“moderate stringency hybridization conditions” (or “moderateconditions”). Exemplary “moderate stringency hybridization conditions”include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDSat 37° C., and a wash in 1×SSC at 45° C. A positive hybridization is atleast twice background. Those of ordinary skill will readily recognizethat alternative hybridization and wash conditions can be utilized toprovide conditions of similar stringency. Nucleic acids which do nothybridize to each other under moderate stringency hybridizationconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, e.g., when a copyof a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

The terms “substantial identity” or “similarity” in the context of apeptide indicates that a peptide comprises a sequence with at least 60%sequence identity to a reference sequence, usually at least 70%,preferably 80%, more preferably 85%, most preferably at least 90% or 95%sequence identity to the reference sequence over a specified comparisonwindow. Preferably, optimal alignment is conducted using the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443.An indication that two peptide sequences are substantially identical isthat one peptide is immunologically reactive with antibodies raisedagainst the second peptide. Thus, a peptide is substantially identicalto a second peptide, for example, where the two peptides differ only bya conservative substitution. Generally, similariy is determined using acomparison window having a length of any number from 20 contiguouspositions to the number of residues in the full-length core regionsequence (i.e., the region of optimal alignment with rSK2 from aminoacid residue 135 to 462), where the comparison window is within the coresequence.

The terms “oligonucleotide” or “polynucleotide” probes include referenceto both double stranded and single stranded DNA or RNA. The terms alsorefer to synthetically or recombinantly derived sequences essentiallyfree of non-nucleic acid contamination.

As used herein, “contact” or “contacting” means to place in directphysical association.

“Biological sample” as used herein is a sample of biological tissue orfluid that contains an IK and/or SK channel protein or nucleic acidencoding the corresponding IK and/or SK channel protein. Such samplesincude, but are not limited to, sputum, amniotic fluid, blood, bloodcells (e.g., white cells), or tissue. Biological samples may alsoinclude sections of tissues such as frozen sections taken forhistological purposes. Examples of biological samples include a cellsample from nervous, muscular, glandular or epithelial tissue or fromthe immune system (e.g., T cells). A biological sample is typicallyobtained from a eukaryotic organism, preferably a multicellulareukaryotes such as insect, protozoa, birds, fish, reptiles, andpreferably a mammal such as rat, mice, cow, dog, guinea pig, or rabbit,and most preferably a primate such as macaques, chimpanzees, or humans.

The term “antibody” also includes antigen binding forms of antibodies(e.g., Fab, F(ab)₂). The term “antibody” refers to a polypeptidesubstantially encoded by an immunoglobulin gene or immunoglobulin genes,or fragments thereof which specifically bind and recognize an analyte(antigen). The recognized immunogiobulin genes include the kappa,lambda, alpha, gamma, delta, epsilon and mu constant region genes, aswell as the myriad immunoglobulin variable region genes. Light chainsare classified as either kappa or lambda. Heavy chains are classified asgamma, mu, alpha, delta, or epsilon, which in turn define theimmunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, eacn pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist e.g., as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H)-C_(H)1 by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region, thereby converting the F(ab)′₂ dimer intoan Fab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see, Fundamental Immunology, Third Edition, W. E. Paul,ed., Raven Press, N.Y. 1993). While various antibody fragments aredefined in terms of the digestion of an intact antibody, one of skillwill appreciate that such fragments may be synthesized de novo eitherchemically or by utilizing recombinant DNA methodology. Thus, the termantibody, as used herein, also includes antibody fragments such assingle chain Fv, chimeric antibodies (i.e., comprising constant andvariable regions from different species), humanized antibodies (i.e.,comprising a complementarity determining region (CDR) from a non-humansource) and heteroconjugate antibodies (e.g., bispecific antibodies).

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine) can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

-   1) Alanine (A), Serine (S), Threonine T;-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and-   6) Phenyialanine (F), Tyrosine (Y), Tryptophan (W).    See also, Creighton (1984) Proteins W. H. Freeman and Company.

The terms “biologically pure” or “isolated” refer to material which issubstantially or essentially free from components which normallyaccompany or interact with it as found in its naturally occurringenvironment. The isolated material optionally comprises material notfound with the material in its natural environment.

The phrase “encodes a protein which could be encoded by a nucleic acidthat selectively hybridizes under moderate stringency hybridizationconditions to a sequence selected from the group consisting of:” in thecontext of nucleic acids refers io those nucleic acids encodingnaturally occurring proteins or derivatives of natural proteins, butwhich are deliberately modified or engineered to no longer hybridize tothe protein of natural origin under the stated conditions.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements which permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed, and a promoter.

The phrase “functional effects” in the context of assays for testingcompounds affecting the channel includes the determination of anyparameter that is indirectly or directly under the influence of thechannel. It includes changes in ion flux and membrane potential but alsoincludes other physiologic effects such increases or decreases oftranscription or hormone release.

By “selectively hybridizing” or “selective hybridization” or“selectively hybridizes” is meant hybridization, under stringenthybridization conditions, of a nucleic acid sequence to a specifiednucleic acid target sequence to a detectably greater degree than itshybridization to nonarget nucleic acid sequences and/or to thesubstantial exclusion of non-target nucleic acids. Selectivelyhybridizing sequences have at least 80% sequence identity, preferably90% sequence identity, and most preferably 100% sequence identity (i.e.,complementary) with each other. “Percentage of sequence identity” isdetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucteotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

The terms “stringent conditions” or “stringent hybridization conditions”refer to conditions under which a probe will hybridize to its targetsequence, to a detectably greater degree than other sequences. Stringentconditions are sequence-dependent and will be different in differentcircumstances. Longer sequences hybridize specifically at highertemperatures. Generally, stringent conditions are selected to be about5° C. lower than the thermal melting point (Tm) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of acomplementary target sequence hybridizes to a perfectly matched probe.Typically, stringent conditions will be those in which the saltconcentration is less than about 1.0 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of30% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 2×SSC at 50° C.Exemplary high stringency conditions include hybridization in 50%formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C.

“Stringent hybridization conditions” or “stringent conditions” in thecontext of nucleic acid hybridization assay formats are sequencedependent, and are different under different environmental parameters.An extensive guide to the hybridization of nucleic acids is found inTijssen (1993) Laboratory Techniques in Biochemistry and MolecuiarBiology—Hybridization with Nucleic Acid Probes Part I, Chapter 2“Overview of principies of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures.

By “hybridization complex” is meant a duplex nucleic acid sequenceformed by selective hybridization of two single-stranded nucleic acidsequences with each other.

By “host cell” is meant a cell which contains an expression vector andsupports the replication or expression of the expression vector. Hostcells may be prokaryotic cells such as E. coli, or eukaryotic cells suchas yeast, insect, amphibian, or mammalian cells.

By “conductance” is meant electrical conductance. Electrical conductanceis conveniently measured in Siemens (1/ohm=mho). Unitary conductance isdetermined by measuring single channel currents using a patch clampprotocol under conditions set forth in Example 6 (i.e., in an oocyte)using a symmetrical potassium ion concentration of 120 mM. Seegenerally, Hille, B., Ionic Channels of Excitable Membranes, 2nd ed.,Sinauer Assoc., Sunderland, Mass. In the context of the presentinvention, “conductance” refers to the unitary electrical conductance ofa single homomeric protein of the referenced SK or IK channel protein.

By “when expressed in an oocyte leads to formation of an SK channel”includes reference to expression of a referenced SK protein in which aplurality of the referenced SK proteins are assembled to form, bythemselves or in conjunction with other endogenous Xenopus oocytemolecules, an SK channel. Expression within a Xenopus oocyte isdisclosed in the Examples provided herein, e.g., Example 3.

By “when expressed in an oocyte leads to formation of acalcium-activated potassium channel” includes reference to expression ofa referenced IK andlor SK protein in which a plurality of the referencedIK and/or SK proteins are assembled to form, by themselves or inconjunction with other endogenous Xenopus oocyte molecules, acalcium-activated potassium channel. Expression within a Xenopus oocyteis disclosed in the Examples provided herein, e.g., Example 3.

By “immunologically reactive conditions” is meant conditions which allowan antibody, generated to a particular epitope, to bind to that epitopeto a detectably greater degree than the antibody binds to substantiallyall other epitopes. Immunologically reactive conditions are dependentupon the format of the antibody binding reaction and typically are thoseutilized in immunoassay protocols. See Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, for a description of immunoassay formats and conditions.

By “antibody reactive to a protein” is meant the protein is“specifically immunoreactive with an antibody.”

The phrase “specifically immunoreactive with an antibody”, or“specifically binds to an antibody” when referring to a protein orpeptide, refers to a binding reaction between an antibody and a proteinhaving an epitope recognized by the antigen binding site of theantibody. This binding reaction is determinative of the presence of aprotein having the recognized epitope amongst the presence of aheterogeneous population of proteins and other biologics. Thus, underdesignated immunoassay conditions, the specified antibodies bind to aprotein having the recognized epitope and bind, if at all, to adetectably lesser degree to other proteins lacking the epitope which arepresent in the sample.

Specific binding to an antibody under such conditions may require anantibody that is selected for its specificity for a particular protein.For example, antibodies raised to the calcium activated potassiumchannel protein with the amino acid sequence depicted in SEQ ID NO:1, 2,3, 4, 19, 20, 30, 32, 43, and 47 can be selected from to obtainantibodies specifically immunoreactive with small and/or intermediatecalcium activated potassium channel proteins and not with otherproteins. The proteins used as immunogens can be in native conformationor denatured so as to provide a linear epitope.

A variety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select monoclonalantibodies specifically immunoreactive with a protein. See Harlow andLane (1988) Antibodies. A Laboratory Manual, Cold Spring HarborPublications, New York, for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity.

By “transfected” is meant the introduction of a nucleic acid into aeukaryotic cell where the nucleic acid may be incorporated into thegenome of the cell (i.e., chromosome, plasmid, or mitochondrial DNA),converted into an autonomous replicon, or transiently expressed (e.g.,transfected mRNA). The transfection can be in vivo or ex vivo. “Ex vivo”means outside the body of the organism from which a cell or cells isobtained or from which a cell line is isolated. Ex vivo transfection ispreferably followed by re-infusion of the cells back into the organism.In contrast, by “in vivo” is meant within the body of the organism fromwhich the cell was obtained or from which a cell line is isolated.

By “antigen” is meant a substance to which an antibody can be generatedand to which the antibody is specifically immunoreactive with. Anantibody immunologically reactive with a particular antigen can begenerated in vivo or by recombinant methods such as selection oflibraries of recombinant antibodies in phage or similar vectors. See,e.g., Huse et al. (1989) Science 246:1275-1281; and Ward, et al. (1989)Nature 341:544-546; and Vaughan et al. (1996) Nature Biotechnology,14:309-314.

By “encoding” or “encoded”, with respect to a specified nucleic acid, ismeant comprising the information for translation into the specifiedprotein. The information is specifled by the use of codons. Typically,the amino acid sequence is encoded by the nucleic acid using the“universal” genetic code. However, variants of the universal code, suchas is present in some plant, animal, and fungal mitochondria, thebacterium Mycoplasma capricolum (Proc. Natl. Acad. Sci., 82:2306-2309(1985), or the ciliate Macronucleus, may be used when the nucleic acidis expressed using these organisms.

By “contiguous amino acids from” in the context of a specified number ofamino acid residues from a specified sequence, is meant a sequence ofamino acids of the specified number from within the specified referencesequence which has the identical order of amino acids each of which isdirectly adjacent to the same amino acids as in the reference sequence.

By “small conductance, calcium activated potassium channel” or “SKchannel” is meant a membrane channel which is not voltage-gated,activated by calcium from about 30 nM to 10 μM, and has a unitaryconductance of from about 2 to 60 pS, often 2 to 25 pS, when measuredunder a symmetrical potassium concentration of 120 mM using theconditions specified in Example 6. An SK channel comprises multiple SKchannel proteins as subunits, typically four SK channel proteins (e.g.,full length or substantially full length SK channel proteins).

By “small conductance, calcium-activated channel protein” or “SK channelprotein” is meant a peptide of at least 10 contiguous amino acids inlength from an amino acid sequence which makes up an SK channel. Theseproteins, when full length, serve as monomers of the SK channel. Thus,an SK channel protein can have the functional characteristics to form aheteromeric or homomeric protein with the functional characteristics ofan SK channel, or be a peptide fragment thereof. For example, bothN-terminal extended rsk3 (SEQ ID NO:43 and truncated rsk3 (SEQ ID NO:3)demonstrate virtually identical functional characteristics.

By “intermediate conductance, calcium-activated potassium channel” or“IK channel” is meant a membrane channel which is not voltage-gated,activated by calcium from about 30 nM to 10 μM, and has in its broadestcontext a unitary inward conductance of from about 20 to 80 pS, but morelikely 30 to 70 pS, 40 to 60 pS, or most preferably about 35 to 40 pSwhen measured under a symmetrical potassium concentration of 120 mMusing the conditions specified in Example 6. An IK channel comprisesmultiple IK channel proteins as subunits, typically four IK channelproteins (e.g., full length or substantially full length IK channelproteins).

By “intermediate conductance, calcium-activated channel protein” or “IKchannel protein” is meant a peptide of at least 10 contiguous aminoacids in length from an amino acid sequence which makes up an IKchannel. These proteins, when full length, serve as monomers of the IKchannel. Thus, an IK channel protein can have the functionalcharacteristics to form a heteromeric or homomeric protein with thefunctional characteristics of an IK channel, or be a peptide fragmentthereof.

By “calcium-activated potassium channel” means a small conductance,calcium-activated potassium (SK) channel, and an intermediateconductance, calcium-activated potassium (IK) channel.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

By “specifically reacts” or “specifically reactive” is meant a reactionof the specificity exhibited by that between an antibody and a proteinwhich “specifically binds” with that antibody.

By “human genomic library” is meant a collection of isolated DNAmolecules which substantially represent the entire genome of a human.Construction of genomic libraries is taught in standard molecularbiology references such as Berger and Kimmel, Guide to Molecular CloningTechniques, Methods in Enzymology volume 152 Academic Press, Inc., SanDiego, Calif. (Berger); Sambrook et al. (1989) Molecular Cloning—ALaboratory Manual (2nd ed.) Vol. 1-3; and Current Protocols in MolecularBiology, F. M. Ausubel et al., eds., Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(1994 Supplement) (Ausubel).

By “amplified” is meant the construction of multiple copies of a nucleicacid sequence or multiple copies complementary to the nucleic acidsequence using at least one of the nucleic acid sequences as a template.Amplification systems include the polymerase chain reaction (PCR)system, ligase chain reaction (LCR) system, nucleic acid sequence basedamplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicasesystems, transcription-based amplification system (TAS), and stranddisplacement amplification (SDA). See, e.g., Diagnostic MolecularMicrobiology: Principles and Applications, Ed. D. H. Persing et al.,American Society for Microbiology, Washington, D.C.

The term “residue” or “amino acid residue” or “amino acid” as usedherein refers to an amino acid that is incorporated into a protein,polypeptide, or peptide (collectively “peptide”). The amino acid may bea naturally occurring amino acid and, unless otherwise limited, mayencompass known analogs of natural amino acids that can function in asimilar manner as naturally occurring amino acids.

By “segment of nucleic acid” is meant a nucleic acid sequence of any oneof from 15 to about 1500 nucleotides, or nucleotide analogs, in lengthor concatamers of such sequence.

By “determining the functional effect” is meant examining the effect ofa compound that increases or decreases potassium ion flux on a cell orcell membrane in terms of cell and cell membrane function. Preferably,the term refes to the functional effect of the compound on SK and IKchannel activity, e.g., changes in conductance, voltage gating and thelike.

Small and Intermediate Conductance, Calcium-Activated Potassium ChannelProteins

The present invention provides intermediate conductance,calcium-activated (IK) potassium channel proteins, and smallconductance, calcium-activated (SK) channel proteins (collectively,“calcium-activated potassium channels”). The isolated small conductance,calcium-activated (SK) channel proteins of the present inventioncomprise at least N amino acids from any one of the sequences selectedfrom the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:43, and SEQ ID NO:47, andconservatively modified variants thereof, where N is any one of theintegers selected from the group consisting of from 10 to 600 and thesequence is unique to the protein of origin.

Similarly, the isolated intermediate conductance, calcium-activated (IK)channel proteins of the present invention comprise at least N aminoacids from SEQ ID NO:32 and conservatively modified variants thereof,where N is any one of the integers selected from the group consisting offrom 10 to 600 and the sequence is unique to the protein of origin.

Typically, the calcium-activated potassium channel proteins and specificpeptides are at least 15, 25, 35, or 50 amino acids in length, morepreferably at least 100, 200, 300, 400, or 500 amino acids in length,and most preferably the full length of SEQ ID NOS:1, 2, 3, 4, 19, 20,32, 43, or 47, or conservatively modified variants thereof. Thus, thepresent invention provides full-length and subsequences of SEQ ID NO:1,2, 3, 4, 19, 20, 32, 43, and 47 and full-length and subsequences ofconservatively modified variants of SEQ ID NO:1, 2, 3, 4, 19, 20, 32,43, and 47. A “full-length” sequence of SEQ ID NO:1, 2, 3, 4, 19, 20,32, 43, or 47 means the sequence of SEQ ID NO:1, 2, 3, 4, 19, 20, 32, 43or 47, respectively. A “full-length” sequence of a conservativelymodified variant of SEQ ID NO:1, 2, 3, 4, 19, 20, 32, 43 or 47 means aconservatively modified variant of SEQ ID NO:1, 2, 3, 4, 19, 20, 32, 43or 47 respectively. The calcium-activated potassium channel proteins andpeptides of the present invention can be used as immunogens for thepreparation of immunodiagnostic probes for assessing increased ordecreased expression of calcium-activated potassium channels in drugscreening assays.

The calcium-activated potassium channel proteins of the presentinvention also include proteins which have substantial identity (i.e.,similarity) to a calcium-activated potassium channel protein of at leastN amino acids from any one of the sequences selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO: 43, and SEQ ID NO: 47 andconservatively modified variants thereof, where N is any one of theintegers selected from the group consisting of 10 to 600. Generally, thecalcium-activated potassium channel proteins are at least 50, typicallyat least 100, preferably at least 200, more preferably at least 300, andmost preferably at least 400 amino acid residues in length. Typically,the substantially similar or conservatively modified variant of thecalcium-activated potassium SK or IK channel protein is a eukaryoticprotein, preferably from a multicellular eukaryotes such as insects,protozoans, birds, fishes, amphibians, reptiles, or mammals.

The SK channel proteins which are substantially identical to, or aconservatively modified variant of, an SK channel protein having asequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:43 and SEQ ID NO:47 willspecifically react, under immunologically reactive conditions, with animmunoglobulin reactive to an SK channel protein selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:43, and SEQ ID NO:47.

Similarly, IK channel proteins which are substantially identical to, ora conservatively modified variant of, an IK channel protein having asequence selected from SEQ ID NO:32 will specifically react, underimmunologically reactive conditions, with an immunoglobulin reactive toan IK channel protein such as SEQ ID NO:32. A variety of immunoassayformats may be used to assess such an immunologically specific reactionincluding, for example, ELISA, competitive immunoassays,radioimmunoassays, Western blots, indirect immunofluorescent assays andthe like.

Altenatively, the SK channel proteins which are substantially identicalto, or are a conservatively modified variant of, an SK channel proteinhaving a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:43, and SEQ ID NO:47will comprise an amino acid sequence which has any one of the valuesfrom 60% to 100% similarity to a comparison window within the coresequence (or “core region”) of an SK channel protein selected from thegroup consisting of SEQ ID NOS:1, 2, 3, 4, 19, 20, 43, and 47. IKchannel proteins which are substantially identical to, or are aconservatively modified variant of, an IK channel protein having thesequence of SEQ ID NO:32 will comprise an amino acid sequence which hasany one of the values from 60% to 100% similarity to a comparison windowwithin the core sequence (or “core region”) of the IK channel proteinhIK1.

Thus, similarity is determined by reference to the core region orsubsequence thereof. The core region of hSK1 (SEQ ID NO:1) is from aminoacid residue 124 through 451 (SEQ ID NO:27). The core region of rSK2(SEQ ID NO:2) is from amino acid residue 135 through 462. The coreregion of truncated rSK3 (SEQ ID NO:3) is from amino acid residue 109through 436. The core region of N-terminal extended rSK3 (SEQ ID NO:43)is from 288-615. The core region of rSK1 (SEQ ID NO:4) is defined by theregion which aligns with the foregoing regions. The core region of hSK2(SEQ ID NO:19) is from amino acid residue 134 through 461. The coreregion of truncated hSK3 (SEQ ID NO:20) is from amino acid residue 109through 436. The core region of N-terminal extended hSK3 (SEQ ID NO:47)is from 238-465. Thus, the core region of SEQ ID NOS:1-4, 19, 20, 43 and47 are inclusive of and defined by the amino acid residue subsequencesLSDYALIFGM (SEQ ID NO:17) at the amino proximal end and QRKFLQAIHQ (SEQID NO:18) at the carboxyl proximal end. The core region of hIK1 (SEQ IDNO:32) is amino acids 25 through 351. A subsequence of the core regionhas a length of any one of the numbers from 10 to the length of a coresequence of SEQ ID NOS: 1, 2, 3, 4, 19, 20, 32, 43 or 47. Preferably, SKor IK channel proteins comprise an amino acid sequence having at least90% similarity over a comparison window of 20 contiguous amino acidsfrom within the core sequence.

Similarity is also determined by reference to functional characteristicsof the calcium activated channel protein. For example, the presentinvention provides several SK3 amino acid sequences, which whenexpressed have virtually identical currents. cDNAs encoding rSK3 havebeen isolated in two different forms. The first, SEQ ID NO:44 encodingSEQ ID NO:43, is the endogenous rSK3 or N-terminal extended rSK3. Thesecond, SEQ ID NO:16, encoding SEQ ID NO:3, is truncated relative to SEQID NO:43 at the N-terminus. Truncated rSK3 protein (SEQ ID NO:3) alsohas a different C-terminus, in which the last 9 amino acids of SEQ IDNO:43 are replaced with 5 different amino acids. Although thesesequences differ at both the N- and C-terminus, they express virtuallyidentical currents. Since the N-terminal extended and truncated SK3express the same current, the N-terminal extension not essential tochannel function per se but is likely involved in targeting the proteinto a specific location in the cell.

Similarly, two cDNAs for hSK3 have been identified: N-terminal extendedhSK3 (SEQ ID NO:48, encoding SEQ ID NO:47) and truncated hSK3 (SEQ IDNO:22, encoding SEQ ID NO:20). In addition, a similar N-terminalextension may exist for SK2. Genomic sequences from the mouse for bothSK2 and SK3 demonstrate that both have an extended open reading frame,which is contiguous with the amino acids sequences for which functionalcurrent expression has been demonstrated. Thus, substantially identicalSK channel proteins, or conservatively modified variants thereof, arealso identified on the basis of functional characteristics.

The present invention provides functional SK and IK channel proteins andsubsequences thereof. Functional SK channels of the present inventionhave a unitary conductance of between 2 and 60 pS, more usually 5 and 25pS, and molecular weights between 40 and 100 Kd for each of the SKchannel proteins which make up the SK channel, more usually 50 to 80 kD.Functional IK channels have a unitary conductance of between 20 and 80pS, and often 30 to 60 pS. Unitary conductance may be convenientlydetermined using inside-out or outside-out patch clamp configurations.These configurations are particularly indicated for the study of thebiophysics of ionic channels (kinetics, conductivity, selectivity,mechanism of permeation and block). Patch clamp methods are well knownin the art. See, e.g., the review of Franciolini, Patch clamp techniqueand biophysical study of membrane channels, Experientia, 42(6):589-594(1986); and Sakmann et al., Patch clamp techniques for studying ionicchannels in excitable membranes, Annual Review of Physiology, 46:455-472(1984).

The isolated SK and IK proteins within the scope of the presentinvention include those which when full-length and expressed in a cellfrom a quiet line, define a functionality and pharmacology indicative ofan SK channel or IK channel, respectively. A quiet line is a cell linethat in its native state (e.g., not expressing recombinant SK or IKchannels) has low or uninteresting electric activity, e.g., a CHO cellline. For example, a control cell (without expression of a putative SKchannel of the present invention) and an experimental cell (expressing aputative SK channel) are maintained under conditions standard formeasurement of electrophysiological paramaters as provided in theworking examples disclosed herein. Each cell is treated with a calciumionophore. Exemplary ionophores include, but are not limited to, suchstandard compounds as ionomycin (Sigma Chemical Co.) or A23187 (SigmaChemical Co.). A cell is often treated with an ionophore at aconcentration of about 1 μM.

Subsequently, electrophysiological measurements of the cells are takento detect induction of a potassium current (e.g., by radiotracer), or achange in conductance of the cell (e.g., by patch clamp), or a change involtage (e.g., by fluorescent dye). If the presence an ion channel isindicated by a calcium induced change, subsequent tests are used tocharacterize the channel as an SK channel of the present invention.Preferably, at least two characteristics are determined, more preferablyat least 3, or 4 are determined. Characteristics of SK channels of thepresent invention are disclosed more fully herein.

For example, a cell expressing an SK channel of the present inventioncan have a conductance of between 2 to 30 pS, often between 2 to 25 pS,can, but not necessarily, exhibit block by apamin at a range from 10 pMto about 100 nM, can comprise an SK channel protein of about 40 to 80kD, can exhibit sequence similarity of at least 60%, and more preferablyat least 70%, 80%, 90% or 95% in an alignment with the core regions ofthe exemplary SK channel proteins disclosed herein, and can bespecifically reactive, under immunologically reactive conditions, withan antibody raised to an exemplary SK or IK channel disclosed herein(e.g., SEQ ID NO:1-4, 19, 20, 32, 43 and 47). Such standard methods aidin the identification of SK proteins of the present invention. Cellsexpressing an IK channel have the same functional characteristics exceptthey are blocked by CTX but not blocked by IBX or apamin and have aunitary conductance of between 20 and 80, often 35 to 40 pS.

Solid phase synthesis of SK or IK channel proteins of less than about 50amino acids in length may be accomplished by attaching the C-terminalamino acid of the sequence to an insoluble support followed bysequential addition of the remaining amino acids in the sequence.Techniques for solid phase synthesis are described by Barany andMerrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides:Analysis, Synthesis, Biology. Vol. 2: Special Methods in PeptideSynthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156(1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed.Pierce Chem. Co., Rockford, Ill. (1984). SK or IK channel proteins ofgreater length may be synthesized by condensation of the amino andcarboxy termini of shorter fragments. Methods of forming peptide bondsby activation of a carboxy terminal end (e.g., by the use of thecoupling reagent N,N′-dicycylohexylcarbodiimide)) is known to those ofskill.

Obtaining Nucleic Acids Encoding Calcium-Activated Potassium ChannelProteins

The present invention provides isolated nucleic acids of RNA, DNA, orchimeras thereof, which encode calcium activated, SK channel proteins(“SK channel protein nucleic acids”) or calcium activated, IK channelproteins (“IK channel protein nucleic acids) as discussed more fullyabove. Nucleic acids of the present invention can be used as probes, forexampie, in detecting deficiencies in the level of mRNA, mutations inthe gene (e.g., substitutions, deletions, or additions), for monitoringupregulation of SK or IK channels in drug screening assays, or forrecombinant expression of SK or IK channel proteins for use asimmunogens in the preparation of antibodies.

Nucleic acids encoding the calcium-activated potassium channel proteinsof the present invention can be made using standard recombinant orsynthetic techniques. With the amino acid sequences of the SK or IKchannel proteins herein provided, one of skill can readily construct avariety of clones containing functionally equivalent nucleic acids, suchas nucleic acids which encode the same protein. Cloning methodologies toaccomplish these ends, and sequencing methods to verify the sequence ofnucleic acids are well known in the art. Examples of appropriate cloningand sequencing techniques, and instructions sufficient to direct personsof skill through many cloning exercises are found in Sambrook, et al.,Molecular Cloning: A Laboratory Manual (2nd Ed., Vols. 1-3, Cold SpringHarbor Laboratory (1989)), Methods in Enzymology, Vol. 152: Guide toMolecular Cloning Techniques (Berger and Kimmel (eds.), San Diego:Academic Press, Inc. (1987)), or Current Protocols in Molecular Biology,(Ausubel, et al. (eds.), Greene Publishing and Wiley-lnterscience, NewYork (1987). Product information from manufacturers of biologicalreagents and experimental equipment also provide information useful inknown biological methods. Such manufacturers include the SIGMA chemicalcompany (Saint Louis, Mo.), R&D systems (Minneapolis, Minn.), PharmaciaLKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories, Inc. (PaloAlto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee,Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc.(Gaithersberg, Md.), Fluka Chemica-Biochemika Analytika (Fluka ChemieAG, Buchs, Switzerland), Invitrogen, San Diego, Calif., and AppliedBiosystems (Foster City, Calif.), as well as many other commercialsources known to one of skill.

1. Isolation of SK and IK Channel Proteins by Nucleic Acid Hybridization

The isolated nucleic acid compositions of this invention, whether RNA,cDNA, genomic DNA, or a hybrid of the various combinations, are isolatedfrom biological sources or synthesized in vitro. Deoxynucleotides can beprepared by any suitable method including, for example, cloning andrestriction of appropriate sequences or direct chemical synthesis bymethods such as the phosphotriester method of Narang et al. Meth.Enzymol. 68: 90-99 (1979); the phosphodiester method of Brown et al.,Meth. Enzymol. 68: 109-151 (1979); the diethylphosphoramidite method ofBeaucage et al., Tetra. Lett., 22: 1859-1862 (1981); the solid phasephosphoramidite triester method described by Beaucage and Caruthers(1981), Tetrahedron Letts., 22(20):1859-1862, e.g., using an automatedsynthesizer, e.g., as described in Needham-VanDevanter et al. (1984)Nucleic Acids Res., 12:6159-6168; and, the solid support method of U.S.Pat. No. 4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This may be converted into double stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill wouldrecognize that while chemical synthesis of DNA is limited to sequencesof about 100 bases, longer sequences may be obtained by the ligation ofshorter sequences.

Nucleic acids encoding an SK channel protein of SEQ ID NO:1 may beobtained by amplification of a human hippocampal cDNA library usingisolated nucleic acid primers having the sequence:ATGCCGGGTCCCCGGGCGGCCTGC (SEQ ID NO:5) and TCACCCGCAGTCCGAGGGGGCCAC (SEQID NO:6). Nucleic acids encoding an SK channel protein of SEQ ID NO:2may be obtained by amplification of a rat brain cDNA library usingisolated nucleic acid primers having the sequence:ATGAGCAGCTGCAGGTACAACGGG (SEQ ID NO:7) and CTAGCTACTCTCAGATGAAGTTGG (SEQID NO:8). Nucleic acids encoding an SK channel protein of SEQ ID NO:43may be obtained by amplification of a rat brain cDNA library usingisolated nucleic acid primers having the sequence:ATGAGCTCCTGCAAATACAGCGGT (SEQ ID NO:9) and TTAGCAACTGCTTGAACTTG (SEQ IDNO:10). Nucleic acids encoding an SK channel protein of SEQ ID NO:4 maybe obtained by amplification of a rat brain cDNA library using isolatednucleic acid primers having the sequence TCAGGGAAGCCCCCGACCGTCAGT (SEQID NO:11) and TCACCCACAGTCTGATGCCGTGGT (SEQ ID NO:12). Nucleic acidsencoding an SK channel protein of SEQ ID NO:19 may be obtained byamplification of a human hippocampal cDNA library using isolated nucieicacid primers having the sequence: ATGAGCAGCTGCAGGTACAACG (SEQ ID NO:23)and CTAGCTACTCTCTGATGAAGTTG (SEQ ID NO:24). Nucleic acids encoding an SKchannel protein of SEQ ID NO:20 (hSK3) may be obtained by amplificationof a human hippocampal cDNA library using isolated nucleic acid primershaving the sequence: ATGAGCTCCTGCAAGTATAGC (SEQ ID NO:25) andTTAGCAACTGCTTGAACTTGTG (SEQ ID NO:26). Nucleic acids encoding the IKchannel protein of SEQ ID NO:32 may be obtained by amplification of ahuman pancreas cDNA library using isolated nucleic acid primer pairshaving the sequence: (SEQ ID NOS:38 and 39) and (SEQ ID NOS:40 and 41).

The isolated nucleic acids of the present invention may be cloned, oramplified by in vitro methods, such as the polymerase chain reaction(PCR), the ligase chain reaction (LCR), the transcription-basedamplification system (TAS), the self-sustained sequence replicationsystem (SSR). A wide variety of cloning and in vitro amplificationmethodologies are well-known to persons of skill. Examples of thesetechniques and instructions sufficient to direct persons of skillthrough many cloning exercises are found in Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology 152 Academic Press,Inc., San Diego, Calif. (Berger); Sambrook et al. (1989) MolecularCloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor Press, N.Y., (Sambrook et al.); CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel); Cashion etal., U.S. Pat. No. 5,017,478; and Carr, European Patent No. 0,246,864.

Examples of techniques sufficient to direct persons of skill through invitro amplification methods are found in Berger, Sambrook, and Ausubel,as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCR ProtocolsA Guide to Methods and Applications (Innis et al. eds) Academic PressInc. San Diego, Calif. (1990) (Innis); Amheim & Levinson (Oct. 1, 1990)C&EN 36-47; The Journal Of NIH Research (1991) 3: 81-94; (Kwoh et al.(1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc.Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem., 35:1826; Landegren et al., (1988) Science, 241: 1077-1080; Van Brunt (1990)Biotechnology, 8: 291-294; Wu and Wallace, (1989) Gene, 4: 560; andBarringer et al. (1990) Gene, 89:117.

Isolated nucleic acids encoding SK channel proteins comprise a nucleicacid sequence encoding an SK channel protein selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:43 and SEQ ID NO:47 and subsequencesthereof. In preferred embodiments, the isolated nucleic acid encoding anSK channel protein is selected from the group consisting of: SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:44 and SEQ ID NO:48 and subsequences thereof.

Isolated nucleic acids encoding IK channel proteins comprise a nucleicacid sequence encoding an IK channel protein such as SEQ ID NO:32, andsubsequences thereof. In preferred embodiments, the isolated nucleicacid encoding an IK channel protein is SEQ ID NO:31 and subsequencesthereof.

In addition to the isolated nucleic acids identified herein, theinvention also includes other isolated nucleic acids encodingcalcium-activated potassium channel proteins which selectivelyhybridize, under stringent conditions, to a nucleic acid encoding aprotein selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQID NO:43 and SEQ ID NO:47, and subsequences thereof. Generally, theisolated nucleic acid encoding a calcium-activated potassium channelprotein of the present invention will hybridize under at least moderatestringency hybridization conditions to a nucleic acid sequence from SEQID NOS: 13, 14, 15, 16, 21, 22, 31, 44, or 48 which encodes the coreregion or subsequence thereof. Altematively, or additionally, theisolated nucleic acid encoding the calcium-activated potassium channelprotein will encode an amino acid sequence of at least 60%, 70%, 80%, or90% similarity over the length of the core region. Conveniently, thenucleic acid encoding a subsequence of the core region is obtained fromSEQ ID NOS: 13, 14, 15, 16, 21, 22, 32, 44, or 48 and is at least anyone of from 15 to 400 nucleotides in length, and generally at least 250or 300 nucleotides in length; preferably the nucleic acid will encodethe entire core sequence. The nucleic acid sequence, or subsequencethereof, encoding the calcium-activated potassium channel proteincomprises at least N′ nucleotides in length, where N′ is any one of theintegers selected from the group consisting of from 18 to 2000. Thus,the nucleic acids of the present invention comprise genomic DNA andnuclear transcripts encoding SK and IK channel proteins.

Where the nucleic acid encoding an SK or IK channel protein is to beused as nucleic acid probes, it is often desirable to label the nucleicacid with detectable labels. The labels may be incorporated by any of anumber of means well known to those of skill in the art. However, in apreferred embodiment, the label is simultaneously incorporated duringthe amplification step in the preparation of the nucleic acids. Thus,for example, polymerase chain reaction (PCR) with labeled primers orlabeled nucleotides will provide a labeled amplification product. Inanother preferred embodiment, transcription amplification using alabeled nucleotide (e.g., fluorescein-labeled UTP and/or CTP)incorporates a label into the transcribed nucleic acids.

Alternatively, a label may be added directly to an original nucleic acidsample (e.g., mRNA, polyA mRNA, cDNA, etc.) or to the amplificationproduct after the amplification is completed. Means of attaching labelsto nucleic acids are well known to those of skill in the art andinclude, for example nick translation or end-labeling (e.g., with alabeled RNA) by phosphorylation of the nucleic acid and subsequentattachment (ligation) of a nucleic acid linker joining the samplenucleic acid to a label (e.g., a fluorophore).

Detectable labels suitable for use in the present invention include anycomposition detectable by spectroscopic, radioisotopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Useful labels in the present invention include biotin for staining withlabeled streptavidin conjugate, magnetic beads, fluorescent dyes (e.g.,fluorescein, texas red, rhodamine, green fluorescent protein, and thelike), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase and others commonly usedin an ELISA), and colorimetric labels such as colloidal gold or coloredglass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted light. Enzymatic labels aretypically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

The probes are used to screen genomic or cDNA libraries from any sourceof interest including specific tissues (e.g., heart, brain, pancreas)and animal source such as rat, human, bird, etc. Screening techniquesare known in the art and are described in the general texts cited abovesuch as in Sambrook and Ausubel.

2. Isolation of SK and IK Channel Proteins by Immunoscreening

In addition to using nucleic acid probes for identifying novel forms ofthe protein claimed herein, it is possible to use antibodies to probeexpression libraries. This is a well known technology. (See Young andDavis, 1982 Efficient isolation of genes using antibody probes Proc.Natl. Acad. Sci., U.S.A. 80:1194-1198.) In general, a cDNA expressionlibrary maybe prepared from commercially available kits or using readilyavailable components. Phage vectors are preferred, but a variety ofother vectors are available for the expression of protein, such vectorsinclude but are not limited to yeast, animal cells and Xenopus oocytes.One selects mRNA from a source that is enriched with the target proteinand creates cDNA which is then ligated into a vector and transformedinto the library host cells for immunoscreening. Screening involvesbinding and visualization of antibodies bound to specific proteins oncells or immobilized on a solid support such as nitrocellulose or nylonmembranes. Positive clones are selected for purification to homogeneityand the isolated cDNA then prepared for expression in the desired hostcells. A good general review of this technology can be found in Methodsof Cell Biology Vol 37 entitled Antibodies in Cell Biology, Ed. D J Asaipp 369-382, 1993.

When choosing to obtain calcium activated channel proteins antibodiesselective for the entire protein or portions can be used. Suitablepeptide sequences include, but are not limited to, GHRRALFEKRKRLSDY (SEQID NO:28), FTDASSRSIGAL (SEQ ID NO:29), and ARKLELTKAEKHVHNFMMDTQLTKR(SEQ ID NO:30) or ARKLELTKAEKHVHNFMMDTQLTK (SEQ ID NO:42).

Nucleic Acid Assays

This invention also provides methods of detecting and/or quantifying SKor IK channel protein expression by assaying for the gene transcript(e.g., nuclear RNA, mRNA). The assay can be for the presence or absenceof the normal gene or gene product, for the presence or absence of anabnormal gene or gene product, or quantification of the transcriptionlevels of normal or abnormal SK or IK channel protein gene product.

In a preferred embodiment, nucleic acid assays are performed with asample of nucleic acid isolated from the organism to be tested. In thesimplest embodiment, such a nucleic acid sample is the total mRNAisolated from a biological sample. The nucleic acid (e.g., eithergenomic DNA or mRNA) may be isolated from the sample according to any ofa number of methods well known to those of skill in the art.

Methods of isolating total DNA or mRNA for use in, inter alia, a nucleicacid assay are well known to those of skill in the art. For example,methods of isolation and purification of nucleic acids are described indetail in Chapter 3 of Laboratory Techniques in Biochemistry andMolecular Biology: Hybridization With Nucleic Acid Probes, Part I.Theory and Nucleic Acid Preparation, P. Tijssen, ed. Elsevier, N.Y.(1993). One of skill will appreciate that where alterations in the copynumber of the gene encoding an SK or IK channel protein is to bedetected genomic DNA is preferably isolated. Conversely, whereexpression levels of a gene or genes are to be detected, preferably RNA(mRNA) is isolated.

Frequently, it is desirable to amplify the nucleic acid sample prior tohybridization. One of skill in the art will appreciate that whateveramplification method is used, if a quantitative result is desired, caremust be taken to use a method that maintains or controls for therelative frequencies of the amplified nucleic acids. Methods of“quantitative” amplification are well known to those of skill in theart. For example, quantitative PCR involves simultaneously co-amplifyinga known quantity of a control sequence using the same primers. Thisprovides an internal standard that may be used to calibrate the PCRreaction. The high density array may then include probes specific to theinternal standard for quantification of the amplified nucleic acid.Detailed protocols for quantitative PCR are provided in PCR Protocols, AGuide to Methods and Applications, Innis et al., Academic Press, Inc.N.Y., (1990).

The method of detecting the presence of a nucleic acid sequence encodingan SK channel protein generally comprises: (a) contacting the biologicalsample, under stringent hybridization conditions, with a nucleic acidprobe comprising a nucleic acid segment which selectively hybridizes toa nucleic acid sequence (target) encoding an SK channel protein selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:43, and SEQ ID NO:47; (b)allowing the probe to specifically hybridize to the nucleic acidencoding an SK channel protein to form a hybridization complex, whereindetection of the hybridization complex is an indication of the presenceof the SK nucleic acid sequence in the sample. Detection of an IKchannel protein is accomplished in a similar fashion using a nucleicacid segment which selectively hybridizes to a nucleic acid sequenceencoding an IK channel protein of SEQ ID NO:32.

The nucleic acid segment of the probe is a subsequence of at least N″contiguous nucleotides in length from a nucleic acid encoding an SKchannel selected from the group consisting of SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:44, and SEQ ID NO:48, and complementary sequences thereof. N″ is anany one of the integers selected from the group consisting of each ofthe integers from 15 to 1500. For detecting the presence of an IKchannel protein the nucleic acid segment is a subsequence of at least N″contiguous nucleotides in length from a nucleic acid encoding an IKchannel of SEQ ID NO:31. “Contiguous nucleotides” from a referencednucleic acid means a sequence of nucleotides having the same order anddirectly adjacent to the same nucleotides (i.e., without additions ordeletions) as in the referenced nucleic acid. Typically, the nucleicacid segment is at least 18 nucleotides in length. The preferred lengthof the nucleic acid probe is from 24 to 200 nucleotides in length.

In particularly preferred embodiments, the nucleic acid segment isderived from a nucleic acid which encodes a core region from a proteinselected from the group consisting of SEQ ID NO:1, 2, 3, 4, 19, 20, 32,43 and 47. Conveniently, the nucleic acid which encodes the core regionis a subsequence of a nucleic acid selected from the group consistingof: SEQ ID NOS: 13, 14, 15, 16, 21, 22, 31, 44, 48, and complementarysequences thereof. Usually, and particularly for cross-specieshybridization, the nucleic acid segment would encode an amino acidsequence from within the core region and will be at least 250nucleotides in length, most preferably will encode the entirety of thecore region, and/or will hybridize to the target sequence under moderatestringency hybridization conditions.

Those of skill will appreciate that nucleic acid sequences of the probewill be chosen so as not to interfere in the selective hybridization ofthe nucleic acid segment to the target. Thus, for example, anyadditional nucleotides attached to the nucleic acid segment willgenerally be chosen so as not to selectively hybridize, under stringentconditions, to the nucleic acid target (potential false negative), norto nucleic acids not encoding an SK or IK channel proteins or peptides(potential false positive). The use of negative and positive controls toensure selectivity and specificity is known to those of skill. Ingeneral, the length of the probe should be kept to the minimum lengthnecessary to achieve the desired results. The length of the nucleic acidencoding an SK or IK channel protein or peptide (i.e., the “SK channelprotein nucleic acid” or “IK channel protein nucleic acid”,respectively) is discussed more fully, supra, but is preferably at least30 nucleotides in length.

A variety of nucleic acid hybridization formats are known to thoseskilled in the art. For example, common formats include sandwich assaysand competition or displacement assays. Hybridization techniques aregenerally described in Berger and Kimmel, (1987), supra.; “Nucleic AcidHybridization, A Practical Approach” (Hames, B. D. and Higgins, S. J.(eds.), IRL Press, 1985, Gall and Pardue, (Proc. Natl. Acad. Sci.,U.S.A. 63:378-383 (1969)); and John, Burnsteil and Jones (Nature,223:582-587 (1969)).

Sandwich assays are commercially useful hybridization assays fordetecting or isolating nucleic acid sequences. Such assays utilize a“capture” nucleic acid covalently immobilized to a solid support and alabelled “signal” nucleic acid in solution. The biological sample willprovide the target nucleic acid. The “capture” nucleic acid probe andthe “signal” nucleic acid probe hybridize with the target nucleic acidto form a “sandwich” hybridization complex. To be effective, the signalnucleic acid cannot hybridize with the capture nucleic acid.

In in situ hybridization, the target nucleic acid is liberated from itscellular surroundings in such as to be available for hybridizationwithin the cell while preserving the cellular morphology for subsequentinterpretation and analysis. The following articles provide an overviewof the art of in situ hybridization: Singer et al., Biotechniques4(3):230-250 (1986); Haase et al., Methods in Virology, Vol. VII, pp.189-226 (1984); Wilkinson, “The theory and practice of in situhybridization” In: In situ Hybridization, Ed. D. G. Wilkinson. IRLPress, Oxford University Press, Oxford; and Nucleic Acid Hybridization:A Practical Approach, Ed. Hames, B. D. and Higgins, S. J., IRL Press(1987).

Typically, labelled signal nucleic acids are used to detecthybridization. Complementary nucleic acids or signal nucleic acids maybe labelled by any one of several methods typically used to detect thepresence of hybridized oligonucleotides. The most common method ofdetection is the use of autoradiography with ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P-labelled probes or the like. Other labels include ligands which bindto labelled antibodies, fluorophores, chemi-luminescent agents, enzymes,and antibodies which can serve as specific binding pair members for alabelled ligand.

The label may also allow for the indirect detection of the hybridizationcomplex. For example, where the label is a hapten or antigen, the samplecan be detected by using antibodies. In these systems, a signal isgenerated by attaching fluorescent or enzyme molecules to the antibodiesor, in some cases, by attachment to a radioactive label. (Tijssen,“Practice and Theory of Enzyme Immunoassays,” Laboratory Techniques inBiochemistry and Molecular Biology” (Burdon, van Knippenberg (eds.),Elsevier, pp. 9-20 (1985)).

The detectable label used in nucleic acids of the present invention maybe incorporated by any of a number of means known to those of skill inthe art, e.g., as discussed supra. Means of detecting such labels arewell known to those of skill in the art.

The sensitivity of the hybridization assays may be enhanced through theuse of a nucleic acid amplification system which multiplies the targetnucleic acid being detected. Examples of such systems include thepolymerase chain reaction (PCR) system and the ligase chain reaction(LCR) system. Other methods known in the art are the nucleic acidsequence based amplification (NASBA, Cangene, Mississauga, Ontario) andQ-Beta Replicase systems.

Those of skill will appreciate that abnormal expression levels orabnormal expression products (e.g., mutated transcripts, truncated ornon-sense proteins) are identified by comparison to normal expressionlevels and normal expression products. Normal levels of expression ornormal expression products can be determined for any particularpopulation, subpopulation, or group of organisms according to standardmethods known to those of skill in the art. Typically this involvesidentifying healthy organisms (i.e., organisms with a functional SK orIK channel protein as indicated by such properties as conductance andcalcium sensitivity) and measuring expression levels of the SK or IKchannel protein gene (as described herein) or sequencing the gene, mRNA,or reverse transcribed cDNA, to obtain typical (normal) sequencevariations. Application of standard statistical methods used inmolecular genetics permits determination of baseline levels ofexpression, and normal gene products as well as significant deviationsfrom such baseline levels.

Nucleic Acid Assay Kits

The nucleic acids of this invention can be included in a kit which canbe used to determine in a biological sample the presence or absence ofthe normal gene or gene product encoding an SK or IK channel of thepresent invention, for the presence or absence of an abnormal gene orgene product encoding an SK or IK channel, or quantification of thetranscription levels of normal or abnormal SK or IK channel protein geneproduct. The kit typically includes a stable preparation of nucleic acidprobes for performing the assay of the present invention. Further, thekit may also include a hybridization solution in either dry or iiquidform for the hybridization of probes to target calcium-activatedpotassium channel proteins or calcium-activated potassium channelprotein nucleic acids of the present invention, a solution for washingand removing undesirable and non-hybridized nucleic acids, a substratefor detecting the hybridization complex, and/or instructions forperforming and interpreting the assay.

Expression of Nucleic Acids

Once the nucleic acids encoding an SK or IK channel protein of thepresent invention are isolated and cloned, one may express the desiredprotein in a recombinantly engineered cell such as bacteria, yeast,insect (especially employing baculoviral vectors), and mammalian cells.A “recombinant protein” is a protein produced using cells that do nothave in their native form an endogenous copy of the DNA able to expressthe protein. The cells produce the recombinant protein because they havebeen genetically altered by the introduction of the appropriate isolatednucleic acid sequence (e.g., a vector comprising an SK or IK channelprotein nucleic acid).

It is expected that those of skill in the art are knowledgeable in thenumerous expression systems available for expression of DNA encoding SKor IK channel proteins. No attempt to describe in detail the variousmethods known for the expression of proteins in prokaryotes oreukaryotes will be made.

In brief summary, the expression of natural or synthetic nucleic acidsencoding calcium-activated potassium channel proteins of the presentinvention will typically be achieved by operably linking the DNA or cDNAto a promoter (which is either constitutive or inducible), followed byincorporation into an expression vector. The vectors can be suitable forreplication and integration in either prokaryotes or eukaryotes. Typicalexpression vectors contain transcription and translation terminators,initiation sequences, and promoters useful for regulation of theexpression of the DNA encoding the SK or IK channel protein. To obtainhigh level expression of a cloned gene, it is desirable to constructexpression vectors which contain, at the minimum, a strong promoter todirect transcription, a ribosome binding site for translationalinitiation, and a transcription/translation terminator. One of skillwould recognize that modifications can be made to an SK or IK channelprotein without diminishing its biological activity. Some modificationsmay be made to facilitate the cloning, expression, or incorporation ofthe targeting molecule into a fusion protein. Such modifications arewell known to those of skill in the art and include, for example, amethionine added at the amino terminus to provide an initiation site, oradditional amino acids (e.g., poly His) placed on either terminus tocreate conveniently located restriction sites or termination codons orpurification sequences.

1. Expression in Prokarvotes

Examples of regulatory regions suitable for this purpose in E. coli arethe promoter and operator region of the E. coli tryptophan biosyntheticpathway as described by Yanofsky, Bacteriol. 158:1018-1024 (1984), andthe leftward promoter of phage lambda (P_(L)) as described by Herskowitzand Hagen, Ann. Rev. Genet., 14:399-445 (1980). The inclusion ofselection markers in DNA vectors transfected in E. coli is also useful.Examples of such markers include genes specifying resistance toampicillin, tetracycline, or chloramphenicol. See, Sambrook, et al. fordetails concerning selection markers for use in E. coli.

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing SK channel proteins are availableusing E. coli, Bacillus sp. and Salmonella (Palva, et al., Gene22:229-235 (1983); Mosbach, et al., Nature 302:543-545 (1983)).

When expressing SK or IK channel proteins in S. typhimurium, one shouldbe aware of the inherent instability of plasmid vectors. To circumventthis, the foreign gene can be incorporated into a nonessential region ofthe host chromosome. This is achieved by first inserting the gene into aplasmid such that it is flanked by regions of DNA homologous to theinsertion site in the Salmonella chromosome. After introduction of theplasmid into the S. typhimurium, the foreign gene is incorporated intothe chromosome by homologous recombination between the flankingsequences and chromosomal DNA.

An example of how this can be achieved is based on the his operon ofSalmonella. Two steps are involved in this process. First, a segment ofthe his operon must be deleted in the Salmonella strain selected as thecarrier. Second, a plasmid carrying the deleted his region downstream ofthe gene encoding the SK or IK channel protein is transfected into thehis Salmonella strain. Integration of both the his sequences and a geneencoding an SK or IK channel protein occurs, resulting in recombinantstrains which can be selected as his⁺.

Detection of the expressed protein is achieved by methods known in theart and include, for example, radioimmunoassays, Western blottingtechniques or immunoprecipitation. Purification from E. coli can beachieved following procedures described in U.S. Pat. No. 4,511,503.

2. Expression in Eukaryotes

A variety of eukaryotic expression systems such as yeast, insect celllines, bird, fish, frog, and mammalian cells, are known to those ofskill in the art. As explained briefly below, SK or IK channel proteinsof the present invention may be expressed in these eukaryotic systems.Expression of SK or IK channels in eukaryotes is particularly preferred.

Synthesis of heterologous proteins in yeast is well known. Methods inYeast Genetics, Sherman, F., et al., Cold Spring Harbor Laboratory,(1982) is a well recognized work describing the various methodsavailable to produce the protein in yeast. Suitable vectors usually haveexpression control sequences, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes, and an origin ofreplication, termination sequences and the like as desired. Forinstance, suitable vectors are described in the literature (Botstein, etal., 1979, Gene, 8:17-24; Broach, et al., 1979, Gene, 8:121-133).

Two procedures are used in transfecting yeast cells. In one case, yeastcells are first converted into protoplasts using zymolyase, lyticase orglusulase, followed by addition of DNA and polyethylene glycol (PEG).The PEG-treated protoplasts are then regenerated in a 3% agar mediumunder selective conditions. Details of this procedure are given in thepapers by J. D. Beggs, 1978, Nature (London), 275:104-109; and Hinnen,A., et al., 1978, Proc. Natl. Acad. Sci. USA, 75:1929-1933. The secondprocedure does not involve removal of the cell wall. Instead the cellsare treated with lithium chloride or acetate and PEG and put onselective plates (Ito, H., et al., 1983, J. Bact., 153:163-168).

The calcium-activated potassium channel proteins of the presentinvention, once expressed, can be isolated from yeast by lysing thecells and applying standard protein isolation techniques to the lysates.The monitoring of the purification process can be accomplished by usingWestern blot techniques or radioimmunoassay of other standardimmunoassay techniques.

The sequences encoding the calcium-activated potassium channel proteinscan also be ligated to various expression vectors for use intransfecting cell cultures of, for instance, mammalian, insect, bird,amphibian, or fish origin. Illustrative of cell cultures useful for theproduction of the peptides are mammalian cells. Mammalian cell systemsoften will be in the form of monolayers of cells although mammalian cellsuspensions may also be used. A number of suitable host cell linescapable of expressing intact proteins have been developed in the art,and include the HEK293, BHK21, and CHO cell lines, and various humancells such as COS cell lines, HeLa cells, myeloma cell lines, Jurkatcells. In some embodiments, Xenopus oocytes are used. Those of skillwill recognize that preferred cell lines for expressing SK or IKchannels substantially lack conductances which compete with thoseprovided by the calcium-activated potassium channels of the presentinvention (i.e., “quiet lines”). Expression vectors for these cells caninclude expression control sequences, such as an origin of replication,a promoter (e.g., the CMV promoter, a HSV tk promoter or pgk(phosphoglycerate kinase) promoter), an enhancer (Queen et al. (1986)Immunol. Rev. 89:49), and necessary processing information sites, suchas ribosome binding sites, RNA splice sites, polyadenylation sites(e.g., an SV40 large T Ag poly A addition site), andtranscriptional-terminator sequences. Other animal cells useful forproduction of SK channel proteins are available, for instance, from theAmerican Type Culture Collection Catalogue of Cell Lines and Hybridomas(7th edition, 1992).

Appropriate vectors for expressing SK or IK channel proteins in insectcells are usually derived from the SF9 baculovirus. Suitable insect celllines include mosquito larvae, silkworm, armyworm, moth and Drosophilacell lines such as a Schneider cell line (See Schneider J. Embryol. Exp.Morphol. 27:353-365 (1987).

As indicated above, the vector, e.g., a plasmid, which is used totransfect the host cell, preferably contains DNA sequences to initiatetranscription and sequences to control the translation of the protein.These sequences are referred to as expression control sequences.

As with yeast, when higher animal host cells are employed,polyadenlyation or transcription terminator sequences from knownmammalian genes need to be incorporated into the vector. An example of aterminator sequence is the polyadenlyation sequence from the bovinegrowth hormone gene. Sequences for accurate splicing of the transcriptmay also be included. An example of a splicing sequence is the VP1intron from SV40 (Sprague, J. et al., 1983, J. Virol 45: 773-781).

Additionally, gene sequences to control replication in the host cell maybe incorporated into the vector such as those found in bovine papillomavirus type-vectors. Saveria-Campo, M., 1985, “Bovine Papilloma virus DNAa Eukaryotic Cloning Vector” in DNA Cloning Vol. II a Practical ApproachEd. D. M. Glover, IRL Press, Arlington, Va. pp. 213-238.

The host cells are competent or rendered competent for transfection byvarious means. There are several well-known methods of introducing DNAinto animal cells. These include: calcium phosphate precipitation,fusion of the recipient cells with bacterial protoplasts containing theDNA, treatment of the recipient cells with liposomes containing the DNA,DEAE dextran, electroporation and micro-injection of the DNA directlyinto the cells. The transfected cells are cultured by means well knownin the art. Biochemical Methods in Cell Culture and Virology, Kuchler,R. J., Dowden, Hutchinson and Ross, Inc., (1977). The expressed proteinsare recovered by well known mechanical, chemical or enzymatic means.

Purification of Extressed Peptides

The SK or IK channel proteins of the present invention which areproduced by recombinant DNA technology may be purified by standardtechniques well known to those of skill in the art. Recombinantlyproduced SK or IK channel proteins can be directly expressed orexpressed as a fusion protein. The recombinant calcium-activatedpotassium channel protein of the present invention is purified by acombination of cell lysis (e.g., sonication) and affinitychromatography. For fusion products, subsequent digestion of the fusionprotein with an appropriate proteolytic enzyme releases the desiredrecombinant calcium-activated potassium channel protein.

The calcium-activated potassium channel proteins of this invention,recombinant or synthetic, may be purified to substantial purity bystandard techniques well known in the art, including selectiveprecipitation with such substances as ammonium sulfate, columnchromatography, immunopurification methods, and others. See, forinstance, R. Scopes, Protein Purification: Principles and Practice,Springer-Verlag: New York (1982); Deutscher, Guide to ProteinPurification, Academic Press, 1990. For example, the proteins of thisinvention may be purified by immunoaffinity columns using antibodiesraised to the SK or IK channel proteins as described herein.

Antibodies to Calcium-Activated Potassium Channel Proteins

Antibodies are raised to the SK or IK channel protein of the presentinvention, including individual, allelic, strain, or species variants,and fragments thereof, both in their naturally occurring (full-length)forms and in recombinant forms. Additionally, antibodies are raised tothese proteins in either their native configurations or in non-nativeconfigurations. Anti-idiotypic antibodies can also be generated. Manymethods of making antibodies are known to persons of skill. Thefollowing discussion is presented as a general overview of thetechniques available; however, one of skill will recognize that manyvariations upon the following methods are known.

A. Antibody Production

A number of immunogens are used to produce antibodies specificallyreactive with an SK or IK channel protein. An isolated recombinant,synthetic, or native SK or IK channel protein of 5 amino acids in lengthor greater, and selected from a subsequence of SEQ ID NO:1, 2, 3, 4, 19,20, 32, 43, or 47 are the preferred immunogens (antigen) for theproduction of monoclonal or polyclonal antibodies. Those of skill willreadily understand that the calcium-activated potassium channel proteinsof the present invention are typically denatured prior to formation ofantibodies for screening expression libraries or other assays in which aputative calcium-activated potassium channel protein of the presentinvention is expressed or denatured in a non-native secondary, tertiary,or quartenary structure. Exemplary proteins for use as immunogensinclude, but are not limited to, GHRRALFEKRKRLSDY (SEQ ID NO:28),FTDASSRSIGAL (SEQ ID NO:29), ARKLELTKAEKHVHNFMMDTQLTKR (SEQ ID NO:30),and ARKLELTKAEKHVHNFMMDTQLTK (SEQ ID NO:42). In one class of preferredembodiments, an immunogenic protein conjugate is also included as animmunogen. Naturally occurring SK or IK channel proteins are also usedeither in pure or impure form.

The SK or IK channel protein is then injected into an animal capable ofproducing antibodies. Either monoclonal or polyclonal antibodies can begenerated for subsequent use in immunoassays to measure the presence andquantity of the calcium-activated potassium channel protein. Methods ofproducing polyclonal antibodies are known to those of skill in the art.In brief, an immunogen (antigen), preferably a purified SK or IK channelprotein, an SK or IK channel protein coupled to an appropriate carrier(eg., GST, keyhole limpet hemanocyanin, etc.), or an SK or IK channelprotein incorporated into an immunization vector such as a recombinantvaccinia virus (see, U.S. Pat. No. 4,722,848) is mixed with an adjuvantand animals are immunized with the mixture. The animal's immune responseto the immunogen preparation is monitored by taking test bleeds anddetermining the titer of reactivity to the calcium-activated potassiumchannel protein of interest. When appropriately high titers of antibodyto the immunogen are obtained, blood is collected from the animal andantisera are prepared. Further fractionation of the antisera to enrichfor antibodies reactive to the SK or IK channel protein is performedwhere desired (see, e.g., Coligan (1991) Current Protocols in ImmunologyWiley/Greene, N.Y.; and Harlow and Lane (1989) Antibodies: A LaboratoryManual Cold Spring Harbor Press, N.Y.).

Antibodies, including binding fragments and single chain recombinantversions thereof, against predetermined fragments of SK or IK channelprotein are raised by immunizing animals, e.g., with conjugates of thefragments with carrier proteins as described above. Typically, theimmunogen of interest is an SK or IK channel protein of at least about 5amino acids, more typically the SK or IK channel protein is 10 aminoacids in length, preferably, 15 amino acids in length and morepreferably the calcium-activated potassium channel protein is 20 aminoacids in length or greater. The peptides are typically coupled to acarrier protein (e.g., as a fusion protein), or are recombinantlyexpressed in an immunization vector. Antigenic determinants on peptidesto which antibodies bind are typically 3 to 10 amino acids in length.

Monoclonal antibodies are prepared from cells secreting the desiredantibody. Monoclonals antibodies are screened for binding to an SK or IKchannel protein from which the immunogen was derived. Specificmonoclonal and polyclonal antibodies will usually bind with a K_(D) ofat least about 0.1 mM, more usually at least about 50 μM. and mostpreferably at least about 1 μM or better.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies arefound in, e.g., Stites et al. (eds.) Basic and Clinical Immunology (4thed.) Lange Medical Publications, Los Altos, Calif., and references citedtherein; Harlow and Lane, Supra; Goding (1986) Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press, New York, N.Y.; andKohler and Milstein (1975) Nature 256: 495-497. Summarized briefly, thismethod proceeds by injecting an animal with an immunogen comprising anSK or IK channel protein. The animal is then sacrificed and cells takenfrom its spleen, which are fused with myeloma cells. The result is ahybrid cell or “hybridoma” that is capable of reproducing in vitro. Thepopulation of hybridomas is then screened to isolate individual clones,each of which secrete a single antibody species to the immunogen. Inthis manner, the individual antibody species obtained are the productsof immortalized and cloned single B cells from the immune animalgenerated in response to a specific site recognized on the immunogenicsubstance.

Alternative methods of immortalization include transfection with EpsteinBarr Virus, oncogenes, or retroviruses, or other methods known in theart. Colonies arising from single immortalized cells are screened forproduction of antibodies of the desired specificity and affinity for theantigen, and yield of the monocional antibodies produced by such cellsis enhanced by various techniques, including injection into theperitoneal cavity of a vertebrate (preferably mammalian) host. The SK orIK channel proteins and antibodies of the present invention are usedwith or without modification, and include chimeric antibodies such ashumanized murine antibodies.

Other suitable techniques involve selection of libraries of recombinantantibodies in phage or similar vectors (see, e.g., Huse et al. (1989)Science 246: 1275-1281; and Ward, et al. (1989) Nature 341: 544-546; andVaughan et al. (1996) Nature Biotechnology, 14: 309-314). Alternatively,high avidity human monoclonal antibodies can be obtained from transgenicmice comprising fragments of the unrearranged human heavy and lightchain Ig loci (i.e., minilocus transgenic mice). Fishwild et al., NatureBiotech., 14:845-851 (1996).

Frequently, the SK or IK channel proteins and antibodies will be labeledby joining, either covalently or non-covalently, a substance whichprovides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionucleotides, enzymes, substrates, cofactors, inhibitors,fluorescent moieties, chemiluminescent moieties, magnetic particles, andthe like. Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366.241. Also, recombinant immunoglobulins may be produced. See,Cabilly, U.S. Pat. No. 4,816,567; and Queen et al. (1989) Proc. Nat'lAcad. Sci. USA 86: 10029-10033.

The antibodies of this invention are also used for affinitychromatography in isolating SK or IK channel proteins. Columns areprepared, e.g., with the antibodies linked to a solid support, e.g.,particles, such as agarose, Sephadex, or the like, where a cell lysateis passed through the column, washed, and treated with increasingconcentrations of a mild denaturant, whereby purified SK or IK channelprotein are released.

The antibodies can be used to screen expression libraries for particularexpression products such as normal or abnormal human SK or IK channelprotein. Usually the antibodies in such a procedure are labeled with amoiety allowing easy detection of presence of antigen by antibodybinding.

Antibodies raised against SK or IK channel protein can also be used toraise anti-idiotypic antibodies. These are useful for detecting ordiagnosing various pathological conditions related to the presence ofthe respective antigens.

B. Human or Humanized (Chimeric) Antibody Production

The anti-SK or anti-IK channel protein antibodies of this invention canalso be administered to a mammal (e.g., a human patient) for therapeuticpurposes (e.g., as targeting molecules when conjugated or fused toeffector molecules such as labels, cytotoxins, enzymes, growth factors,drugs, etc.). Antibodies administered to an organism other than thespecies in which they are raised are often immunogenic. Thus, forexample, murine antibodies administered to a human often induce animmunologic response against the antibody (e.g., the human anti-mouseantibody (HAMA) response) on multiple administrations. The immunogenicproperties of the antibody are reduced by altering portions, or all, ofthe antibody into characteristically human sequences thereby producingchimeric or human antibodies, respectively.

i) Humanized (Chimeric) Antibodies

Humanized (chimeric) antibodies are immunoglobulin molecules comprisinga human and non-human portion. More specifically, the antigen combiningregion (or variable region) of a humanized chimeric antibody is derivedfrom a non-human source (e.g., murine) and the constant region of thechimeric antibody (which confers biological effector function to theimmunoglobulin) is derived from a human source. The humanized chimericantibody should have the antigen binding specificity of the non-humanantibody molecule and the effector function conferred by the humanantibody molecule. A large number of methods of generating chimericantibodies are well known to those of skill in the art (see, e.g., U.S.Pat. Nos: 5,502,167, 5,500,362, 5,491,088, 5,482,856, 5,472,693,5,354,847, 5,292,867, 5,231,026, 5,204,244, 5,202,238, 5,169,939,5,081,235, 5,075,431, and 4,975,369). Detailed methods for preparationof chimeric (humanized) antibodies can be found in U.S. Pat. No.5,482.856.

ii) Human Antibodies

In another embodiment, this invention provides for fully human anti-SKchannel protein antibodies. Human antibodies consist entirely ofcharacteristically human polypeptide sequences. The human anti-SK oranti-IK channel protein antibodies of this invention can be produced inusing a wide variety of methods (see, e.g., Larrick et al., U.S. Pat.No. 5,001,065, for review).

In preferred embodiments, the human anti-SK channel protein antibodiesof the present invention are usually produced initially in trioma cells.Genes encoding the antibodies are then cloned and expressed in othercells, particularly, nonhuman mammalian cells. The general approach forproducing human antibodies by trioma technology has been described byOstberg et al. (1983), Hybridoma 2: 361-367, Ostberg, U.S. Pat. No.4,634,664, and Engelman et al., U.S. Pat. No. 4,634,666. Theantibody-producing cell lines obtained by this method are called triomasbecause they are descended from three cells; two human and one mouse.Triomas have been found to produce antibody more stably than ordinaryhybridomas made from human cells.

The genes encoding the heavy and light chains of immunoglobulinssecreted by trioma cell lines are cloned according to methods, includingthe polymerase chain reaction, known in the art (see, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor, N.Y., 1989; Berger & Kimmel, Methods in Enzymology, Vol 152:Guide to Molecular Cloning Techniques, Academic Press, Inc., San Diego,Calif., 1987; Co et al. (1992) J. Immunol, 148: 1149). For example,genes encoding heavy and light chains are cloned from a trioma's genomicDNA or cDNA produced by reverse transcription of the trioma's RNA.Cloning is accomplished by conventional techniques including the use ofPCR primers that hybridize to the sequences flanking or overlapping thegenes, or segments of genes, to be cloned.

Calcium-Activated Potassium Channel Protein Immunoassays

Immunoassays for SK and IK channel proteins can be used for at least twodifferent purposes. They can be used to determine the relatedness of theprotein by virtue of their being able to cross-react immunologically orfor detection of the presence or absense of the channel proteins.

When determining if an unknown protein is related to the channelproteins of this invention, a variety of assays can be used. For exampleand preferred is a competitive immunoassay to test for cross-reactivity.For example, the protein of SEQ ID NO:2 or 32 can be immobilized to asolid support. Proteins or peptides are added to the assay which competewith the binding of the antisera to the immobilized antigen. The abilityof the above proteins to compete with the binding of the antisera to theimmobilized protein is compared to the protein thought to be related tothe test protein.

To assure that the antisera being tested is specific or selectivelybinding to a particular protein, it will be tested for cross-reactivityto other closely related proteins. This allows for the production ofsera that will distinguish between small, intermediate and largeconductance channels. The percent crossreactivity for the above proteinscan be calculated, using standard calculations. Those antisera with lessthan 10% crossreactivity with each of the proteins listed above areselected and pooled. The cross-reacting antibodies are optionallyremoved from the pooled antisera by immunoabsorption with theabove-listed proteins.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein tothe claimed or prototype immunogen protein. In order to make thiscomparison, the two proteins are each assayed at a wide range ofconcentrations and the amount of each protein required to inhibit 50% ofthe binding of the antisera to the immobilized protein is determined. Ifthe amount of protein required is less than twice the amount of theprototype protein, then the second protein is said to specifically bindto an antibody generated to the prototype immunogen. Where theantibodies are generated to a short peptide, the test proteins areoptionally denatured to fully test for selective binding. In situationswhere the target peptide is not readily accessible to the antibodiesbecause the target peptide is part of a larger protein, it is proper tomeasure the relatedness of test proteins against prototype proteins ofsimilar size, e.g., one would test a full length monomer against aprototype, full length monomer even though the antisera was generatedagainst a peptide of the prototype monomer. This simplifies the readingof the test results and avoids having to take into accountconformational problems and molecular weight/molar concentrations in thedetermination of the data generated from the competitive immunoassays.

Means of detecting the SK or IK channel proteins of the presentinvention are not critical aspects of the present invention. In apreferred embodiment, the SK or IK channel proteins are detected and/orquantified using any of a number of well recognized immunologicalbinding assays (see, eg., U.S. Pat. Nos. 4,366,241; 4,376,110;4,517,288; and 4,837,168). For a review of the general immunoassays, seealso Methods in Cell Biology Volume 37: Antibodies in Cell Biology,Asai, ed. Academic Press, Inc. New York (1993); Basic and ClinicalImmunology 7th Edition, Stites & Terr, eds. (1991). Immunologicalbinding assays (or immunoassays) typically utilize a “capture agent” tospecifically bind to and often immobilize the analyte (in this case acalcium-activated potassium channel protein). The capture agent is amoiety that specifically binds to the analyte. In a preferredembodiment, the capture agent is an antibody that specifically binds acalcium-activated potassium channel protein(s) of the present invention.The antibody (anti-SK or anti-IK channel protein antibody) may beproduced by any of a number of means known to those of skill in the artas described herein.

Immunoassays also often utilize a labeling agent to specifically bind toand label the binding complex formed by the capture agent and theanalyte. The labeling agent may itself be one of the moieties comprisingthe antibodylanalyte complex. Thus, the labeling agent may be a labeledSK or IK channel protein or a labeled anti-SK or anti-IK channel proteinantibody. Alternatively, the labeling agent may be a third moiety, suchas another antibody, that specifically binds to the antibody/SK orantibody/IK channel protein complex.

In a preferred embodiment, the labeling agent is a second SK or IKchannel protein antibody bearing a label. Alternatively, the second SKor IK channel protein antibody may lack a label, but it may, in turn, bebound by a labeled third antibody specific to antibodies of the speciesfrom which the second antibody is derived. The second can be modifiedwith a detectable moiety, such as biotin, to which a third labeledmolecule can specifically bind, such as enzyme-labeled streptavidin.

Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelagent. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,generally Kronval, et al. (1973) J. Immunol., 111: 1401-1406, andAkerstrom, et al. (1985) J. Immunol., 135: 2589-2542).

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,analyte, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

While the details of the immunoassays of the present invention may varywith the particular format employed, the method of detecting an SK or IKchannel protein in a biological sample generally comprises the steps ofcontacting the biological sample with an antibody which specificallyreacts, under immunologically reactive conditions, to the SK or IKchannel protein. The antibody is allowed to bind to the SK or IK channelprotein under immunologically reactive conditions, and the presence ofthe bound antibody is detected directly or indirectly.

A. Non-Competitive Assay Formats

Immunoassays for detecting SK or IK channel proteins of the presentinvention include competitive and noncompetitive formats. Noncompetitiveimmunoassays are assays in which the amount of captured analyte (in thiscase an SK or IK channel protein) is directly measured. In one preferred“sandwich” assay, for example, the capture agent (anti-SK or anti-IKchannel protein antibodies) can be bound directly to a solid substratewhere they are immobilized. These immobilized antibodies then capture SKor IK channel protein presentin the test sample. The SK or IK channelprotein thus immobilized is then bound by a labeling agent, such as asecond human SK or IK channel protein antibody bearing a label.Alternatively, the second SK or IK channel protein antibody may lack alabel, but it may, in turn, be bound by a labeled third antibodyspecific to antibodies of the species from which the second antibody isderived. The second can be modified with a detectable moiety, such asbiotin, to which a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

B. Competitive Assay Formats

In competitive assays, the amount of analyte (SK or IK channel protein)present in the sample is measured indirectly by measuring the amount ofan added (exogenous) analyte (SK or IK channel protein) displaced (orcompeted away) from a capture agent (anti-SK or anti-IK channel proteinantibody) by the analyte present in the sample. In one competitiveassay, a known amount of, in this case, SK or IK channel protein isadded to the sample and the sample is then contacted with a captureagent, in this case an antibody that specifically binds the SK or IKchannel protein. The amount of SK or IK channel protein bound to theantibody is inversely proportional to the concentration of SK or IKchannel protein present in the sample.

In a particularly preferred embodiment, the antibody is immobilized on asolid substrate. The amount of SK or IK channel protein bound to theantibody may be determined either by measuring the amount of SK or IKchannel protein present in the corresponding SK or IK channelprotein/antibody complex, or alternatively by measuring the amount ofremaining uncomplexed SK or IK channel protein. The amount of SK or IKchannel protein may be detected by providing a labeled SK or IK channelprotein molecule.

A hapten inhibition assay is another preferred competitive assay. Inthis assay a known analyte, in this case the SK or IK channel protein isimmobilized on a solid substrate. A known amount of anti-SK or anti-IKchannel protein antibody, respectively, is added to the sample, and thesample is then contacted with the immobilized SK or IK channel protein.In this case, the amount of anti-SK or anti-IK channel protein antibodybound to the immobilized SK or IK channel protein is inverselyproportional to the amount of SK or IK channel protein present in thesample. Again the amount of immobilized antibody may be detected bydetecting either the immobilized fraction of antibody or the fraction ofthe antibody that remains in solution. Detection may be direct where theantibody is labeled or indirect by the subsequent addition of a labeledmoiety that specifically binds to the antibody as described above.

C. Other Assay Formats

In a particularly preferred embodiment, Western blot (immunoblot)analysis is used to detect and quantify the presence of an SK or IKchannel protein in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind SK channel protein. The anti-SK or anti-IKchannel protein antibodies specifically bind to the SK or IK channelproteins, respectively, on the solid support. These antibodies may bedirectly labeled or alternatively may be subsequently detected usinglabeled antibodies (e.g., labeled sheep anti-mouse antibodies) thatspecifically bind to the anti-SK or anti-IK channel protein.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see, Monroe et al.(1986) Amer. Clin. Prod. Rev. 5:34-41).

D. Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, so long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, radioisotopic, electrical, optical or chemical means.Useful labels in the present invention include those used in labeling ofnucleic acids as discussed, supra.

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to an anti-ligand (e.g., streptavidin) moleculewhich is either inherently detectable or covalently bound to a signalsystem, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be used in conjunction with the labeled,naturally occurring anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidoreductases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, eg., luminol. For a review of variouslabeling or signal producing systems which may be used, see, U.S. Pat.No. 4,391,904).

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple calorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

Immunoassay Detection Kits

The present invention also provides-for kits for the diagnosis oforganisms (e.g., patients) with a deficiency in the levels of expressedSK or IK channel protein. The kits preferably include one or morereagents for detecting an the amount of SK or IK channel protein in amammal. Preferred reagents include antibodies that specifically bind tonormal SK or IK channel proteins or subsequences thereof. The antibodymay be free or immobilized on a solid support such as a test tube, amicrowell plate, a dipstick and the like. The kit may also containinstructional materials teaching the use of the antibody in an assay forthe detection of SK or IK channel protein. The kit may containappropriate reagents for detection of labels, positive and negativecontrols, washing solutions, dilution buffers and the like.

Assays for Compounds that Increase or Decrease K⁺ Flux

Isolated SK or IK channel nucleic acids of the present invention whichare expressed in cells can be used in a variety of assays to detectcompounds that increase or decrease the flux (i.e., influx or efflux) ofpotassium through the SK or IK channels, respectively. Generally,compounds that decrease potassium ion flux will cause a decrease by atleast 10% or 20%, more preferably by at least 30%, 40%, or 50%, and mostpreferably by at least 70%, 80%, 90% or 100%. Compounds that increasethe flux of potassium ions will cause a detectable increase in thepotassium ion current density by increasing the probability of a SK orIK channel being open and allowing the passage of potassium ions.Typically the flux will increase by at least 20%, 50%, 100%, or 200%,often by at least 400%, 600%, 1,000%, 5,000% or 10,000%. Increased ordecreased flux of potassium may be assessed by determining changes inpolarization (i.e., electrical potential) of the cell expressing the SKor IK channel. A particularly preferred means to determine changes incellular polarization is the voltage-clamp technique. Whole cellcurrents are conveniently determined using the conditions set forth inExample 3. Other known assays include: radiolabeled rubidium flux assaysand fluorescence assays using voltage-sensitive dyes. See, e.g.,Vestergarrd-Bogind et al., J. Membrane Biol., 88:67-75 (1988); Daniel etal., J. Pharmacol. Meth., 25:185-193 (1991); Holevinsky et al., J.Membrane Biology, 137:59-70 (1994). Assays for compounds capable ofinhibiting or increasing potassium flux through the SK channel proteincan be performed by application of the compounds to a bath solution incontact with and comprising cells having an SK or IK channel of thepresent invention. See, e.g., Blatz et al., Nature, 323:718-720 (1986);Park, J. Physiol., 481:555-570 (1994). Generally, the compounds to betested are present in the range from 1 pM to 100 mM. Changes in functionof the channels can be measured in the electrical currents or ionicflux, or by the consequences of changes in currents and flux.

The effects of the test compounds upon the function of the channels canbe measured by changes in the electrical currents or ionic flux or bythe consequences of changes in currents and flux. Changes in electricalcurrent or ionic flux are measured by either increases or decreases influx of cations such as potassium or rubidium ions. The cations can bemeasured in a variety of standard ways. They can be measured directly byconcentration changes of the ions or indirectly by membrane potential orby radiolabeling of the ions. Consequences of the test compound on ionflux can be quite varied. Accordingly, any suitable physiological changecan be used to assess the influence of a test compound on the channelsof this invention. Changes in channel function can be measured by liganddisplacement such as CTX release. When the functional consequences aredetermined using intact cells or animals, one can also measure a varietyof effects such as transmitter release (e.g., dopamine), hormone release(e.g., insulin), transcriptional changes to both known anduncharacterized genetic markers (e.g., northern blots), cell volumechanges (e.g., in red blood cells), immuno-responses (e.g., T cellactivation), changes in cell metabolism such as cell growth or pHchanges.

Preferably, the SK channel of the assay will be selected from a channelprotein of SEQ ID NOS:1, 2, 3, 4, 19, 20, 43 or 47 or conservativelymodified variant thereof. An IK channel of the assay will preferablyhave a sequence as shown in SEQ ID NO:32, or conservatively modifiedvariant thereof. Alternatively, the SK channel of the assay will bederived from a eukaryote and include an amino acid subsequence havingsequence similarity to the core region of SK channel proteins of SEQ IDNOS:1 through 4, 19, 20, 43 and/or 47. The IK will typically be derivedfrom a eukaryote and include an amino acid subsequence having sequencesimilarity to the core region of IK channel proteins of SEQ ID NO:32.Generally, the functional SK or IK channel protein will be at least 400,450, 500, or 550 amino acids in length. The percentage of sequencesimilarity with the core region of a protein selected from the groupconsisting of: SEQ ID NO:1, 2, 3, 4, 19, 20, 32, 43 and 47 will be anyone of the integers between 60 and 100. Generally, the sequencesimilarity will be at least 60%, typically at least 70%, generally atleast 75%, preferably at least 80%, more preferably at least 85%, mostpreferably at least 90%, and often at least 95%. Thus, SK channelhomologs will hybridize, under moderate hybridization conditions, to anucleic acid of at least 300 nucleotides in length from the core regionof a nucleic acid selected from the group consisting of SEQ ID NOS:13,14, 15, 16, 21, 22, 44 and 48 and complementary sequences thereof. IKchannel homologs will hybridize, under moderate hybridizationconditions, to a nucleic acid of at least 300 nucleotides in length fromthe core region of a nucleic acid such as SEO ID NO:31.

The “core region” or “core sequence” of SEQ ID NOS:13-16, 21, 22, 44 and48 corresponds to the encoded region of alignment between SEQ ID NOS: 1,2, 3, 4, 19, 20, 43, and 47 with and from rSK2 (SEQ ID NO:2) amino acidresidue 135 to 462. The core region of hIK1 is from amino acid residue25 through residue 351. In preferred embodiments, the SK channel willhave at least 90% sequence similarity, as compared to the core sequencefrom a sequence of ID NO: 1, 2, 3, 4, 19, 20, 43, or 47 over acomparison window of any of from any one of 20 contiguous amino acidresidues to 300 contiguous amino acid residues from within the coreregion. In preferred embodiments, the IK channel will have at least 90%sequence similarity, as compared to the core sequence of SEQ ID NO:32,over a comparison window of any of from any one of 20 contiguous aminoacid residues to 300 contiguous amino acid residues from within the coreregion.

The SK channel homologs will generally have substantially similarconductance characteristics (e.g., 2-60 pS) and calcium sensitivities(30 nM-10 μM). IK channel homologs will likewise have similar SKchannels conductance characteristics as a IK channel (e.g., 20-80 pS)and calcium sensitivities (30 nM-10 μM). Chimeras formed by expressionof at least two of SEQ ID NOS:1, 2, 3, 4, 19, 20, 32, 43 or 47 can alsobe used; In a preferred embodiment, the cell placed in contact with acompound which is assayed for increasing or decreasing potassium flux isa eukaryotic cell, more preferably an oocyte of Xenopus (e.g., Xenopuslaevis).

Yet another assay for compounds that increase or decrease potassium fluxin calcium activated potassium channels involves “virtual genetics,” inwhich a computer system is used to generate a three-dimensionalstructure of SK and IK proteins based on the structural informationencoded by the amino acid sequence. The amino acid sequence interactsdirectly and actively with a preestablished algorithm in a computerprogram to yield secondary, tertiary, and quaternary structural modelsof the protein. The models of the protein structure are then examined toidentify regions of the structure that have the ability to bind toligands. These regions are then used to identify ligands that bind tothe protein.

The three-dimensional structural model of the protein is generated byinputting channel protein amino acid sequences or nucleic acid sequencesencoding a channel protein into the computer system. The amino acidsequence of the channel protein is selected from the group consistingof: SEQ ID NOS: 1, 2, 3, 4, 19, 20, 32, 43, 47, and conservativelymodified versions thereof. The amino acid sequence represents theprimary sequence of the protein, which encodes the structuralinformation of the protein. The amino acid sequence is input into thecomputer system from computer readable substrates that include, but arenot limited to, electronic storage media (e.g., magnetic diskettes,tapes, cartridges, and chips), optical media (e.g., CD ROM, telephonelines), addresses to internet sites, and RAM. The three-dimensionalstructural model of the channel protein is then generated by theinteraction of the amino acid sequence and the computer system. Thesoftware is commercially available programs such as Biopolymer, Quanta,and Insight.

The amino acid sequence represents a primary structure that encodes theinformation necessary to form the secondary, tertiary and quaternarystructure of the protein. The software looks at certain parametersencoded by the primary sequence to generate the structural model. Theseparameters are referred to as “energy terms,” and primarily includeelectrostatic potential, hydrohobic potential, solvent accessiblesurface, and hydrogen bonding. Secondary energy terms include van derWaals potential. Biological molecules form the structures that minimizethe energy terms in a cumulative fashion. The computer program istherefore using these terms encoded by the primary structure or aminoacid sequence to create the secondary structural model.

The tertiary structure of the protein encoded by the secondary structureis then formed on the basis of the energy terms of the secondarystructure. The user at this point can input additional variables such aswhether the protein is membrane bound or soluble, its location in thebody, and whether it is cytoplasmic, surface, or nuclear. Thesevariables along with the energy terms of the secondary structure areused to form the model of the teritary structure. In modeling thetertiary structure, the computer program matches hydrophobic proteinfaces of secondary structure with like, and hydrophilic secondarystructure with like.

Finally, quaternary structure of multi-subunit proteins can be modeledin a similar fashion, using anisotrophy terms. These terms interfacedifferent protein subunits to energetically minize the interaction ofthe subunits. In the case of channel proteins, typically four identicalsubunits make up the quatemary structure of the channel.

Once the structure has been generated, potential ligand binding regionsare identified by the computer system. Three-dimensional structures forpotential ligands are generated by inputting amino acid and nucleotidesequences or chemical formulas of compounds, as described above. Thethree-dimensional structure of the potential ligand is then compared tothat of the channel protein to identify ligands that bind to the channelprotein. Binding affinity between the protein and ligands is determinedusing energy terms to determine which ligands have an enhancedprobability of binding to the protein.

Computer systems are also used to screen for mutations of SK and IKgenes. Such mutations can be associated with disease states. Once themutations are identified, diagnostic assays can be used to identifypatients having such mutated genes associated with disease states.Identification of the mutated SK and IK genes involves receiving inputof a first nucleic acid sequence encoding a calcium channel proteinhaving an amino acid sequence selected from the group consisting of SEQID NOS:1, 2, 3, 4, 20, 32, 43, 47, and conservatively modified versionsthereof. The sequence is input into the compter system as describedabove. The first nucleic acid sequence is then compared to a secondnucleic acid sequence that has substantial identity to the first nucleicacid sequence. The second nucleic acid sequence is input into thecomputer system in the manner described above. Once the first andsequence sequences are compared, nucleotide differences between thesequences are identified. Such sequences can represent allelicdifferences in SK and IK genes, and mutations associated with diseasestates.

Cellular Transfection and Gene Therapy

The present invention provides packageable SK and IK channel proteinnucleic acids (cDNAs), supra, for the transfection of cells in vitro andin vivo. These packageable nucleic acids can be inserted into any of anumber of well known vectors for the transfection of target cells andorganisms as described below. The nucleic acids are transfected intocells, ex vivo or in vivo, through the interaction of the vector and thetarget cell. The SK or IK channel protein nucleic acid, under thecontrol of a promoter, then expresses the calcium-activated potassiumchannel protein of the present invention thereby mitigating the effectsof absent, partial inactivation, or abnormal expression of the SK or IKchannel protein gene.

Such gene therapy procedures have been used to correct acquired andinherited genetic defects, cancer, and viral infection in a number ofcontexts. The ability to express artificial genes in humans facilitatesthe prevention and/or cure of many important human diseases, includingmany diseases which are not amenable to treatment by other therapies. Asan example, in vivo expression of cholesterol-regulating genes, geneswhich selectively block the replication of HIV, and tumor-suppressinggenes in human patients dramatically improves the treatment of heartdisease, AIDS, and cancer, respectively. For a review of gene therapyprocedures, see Anderson, Science (1992) 256:808-813; Nabel and Felgner(1993),TIBTECH 11: 211-217; Mitani and Caskey (1993) TIBTECH 11:162-166; Mulligan (1993) Science 926-932; Dillon (1993) TIBTECH 11:167-175; Miller (1992) Nature 357: 455-460; Van Brunt (1988)Biotechnology 6(10): 1149-1154; Vigne (1995) Restorative Neurology andNeuroscience 8: 35-36; Kremer and Perricaudet (1995) British MedicalBulletin 51(1) 31-44; Haddada et al. (1995) in Current Topics inMicrobiology and Immunology Doerfler and Böhm (eds) Springer-Verlag,Heidelberg Germany; and Yu et al., Gene Therapy (1994) 1:13-26.

Delivery of the gene or genetic material into the cell is the firstcritical step in gene therapy treatment of disease. A large number ofdelivery methods are well known to those of skill in the art. Suchmethods include, for example liposome-based gene delivery (Debs and Zhu(1993) WO 93/24640; Mannino and Gould-Fogerite (1988) Bio Techniques6(7): 682-691; Rose U.S. Pat No. 5,279,833; Brigham (1991) WO 91/06309;and Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414), andreplication-defective retroviral vectors harboring a therapeuticpolynucleotide sequence as part of the retroviral genome (see, e.g.,Miller et al. (1990) Mol. Cell. Biol. 10:4239 (1990); Kolberg (1992) J.NIH Res. 4:43, and Cornetta et al. Hum. Gene Ther. 2:215 (1991)). Widelyused retroviral vectors include those based upon murine leukemia virus(MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus(SIV), human immuno deficiency virus (HIV), and combinations thereof.See, e.g., Buchscher et al. (1992) J. Virol. 66(5) 2731-2739; Johann etal. (1992) J. Virol. 66 (5):1635-1640 (1992); Sommerfelt et al., (1990)Virol. 176:58-59; Wilson et al. (1989) J. Virol. 63:2374-2378; Miller etal., J. Virol. 65:2220-2224 (1991); Wong-Staal et al., PCT/US94/05700,and Rosenburg and Fauci (1993) in Fundamental Immunology, Third EditionPaul (ed) Raven Press, Ltd., New York and the references therein, and Yuet al., Gene Therapy (1994) supra).

AAV-based vectors are also used to transduce cells with target nucleicacids, e.g., in the in vitro production of nucleic acids and peptides,and in in vivo and ex vivo gene therapy procedures. See, West et al.(1987) Virology 160:38-47; Carter et al. (1989) U.S. Pat. No. 4,797,368,Carter et al. WO 93/24641 (1993); Kotin (1994) Human Gene Therapy5:793-801; Muzyczka (1994) J. Clin. Invst. 94:1351 and Samulski (supra)for an overview of AAV vectors. Construction of recombinant AAV vectorsare described in a number of publications, including Lebkowski, U.S.Pat. No. 5,173,414; Tratschin et al. (1985) Mol. Cell. Biol.5(11):3251-3260; Tratschin, et al. (1984) Mol. Cell. Biol., 4:2072-2081;Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA, 81:6466-6470;McLaughlin et al. (1988) and Samulski et al. (1989) J. Virol.,63:03822-3828. Cell lines that can be transfected by rAAV include thosedescribed in Lebkowski et al. (1988) Mol. Cell. Biol., 8:3988-3996.

A. Ex vivo Transfection of Cells

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with an SK orIK channel protein nucleic acid (gene or cDNA), and re-infused back intothe subject organism (e.g., patient). Various cell types suitable for exvivo transfection are well known to those of skill in the art (see,e.g., Freshney et al., Culture of Animal Cells, a Manual of BasicTechnique, third edition Wiley-Liss, New York (1994)) and the referencescited therein for a discussion of how to isolate and culture cells frompatients).

As indicated above, in a preferred embodiment, the packageable nucleicacid which encodes an SK or IK channel protein is under the control ofan activated or constitutive promoter. The transfected cell(s) express afunctional SK or IK channel protein which mitigates the effects ofdeficient or abnormal SK or IK channel protein gene expression.

In one particularly preferred embodiment, stem cells are used in ex-vivoprocedures for cell transfection and gene therapy. The advantage tousing stem cells is that they can be differentiated into other celltypes in vitro, or can be introduced into a mammal (such as the donor ofthe cells) where they will engraft in the bone marrow. Methods fordifferentiating CD34⁺ cells in vitro into clinically important immunecell types using cytokines such a GM-CSF, IFN-γ and TNF-α are known(see, Inaba et al. (1992) J. Exp. Med. 176, 1693-1702, and Szabolcs etal. (1995) 154: 5851-5861).

Stem cells are isolated for transduction and differentiation using knownmethods. For example, in mice, bone marrow cells are isolated bysacrificing the mouse and cutting the leg bones with a pair of scissors.Stem cells are isolated from bone marrow cells by panning the bonemarrow cells with antibodies which bind unwanted cells, such as CD4⁺ andCD8⁺ (T cells), CD45⁺ (panB cells), GR-1 (granulocytes), and Ia^(d)(differentiated antigen presenting cells). For an example of thisprotocol see, Inaba et al. (1992) J. Exp. Med. 176, 1693-1702.

In humans, bone marrow aspirations from iliac crests are performed e.g.,under general anesthesia in the operating room. The bone marrowaspirations is approximately 1,000 ml in quantity and is collected fromthe posterior iliac bones and crests. If the total number of cellscollected is less than about 2×10⁸/kg, a second aspiration using thesternum and anterior iliac crests in addition to posterior crests isperformed. During the operation, two units of irradiated packed redcells are administered to replace the volume of marrow taken by theaspiration. Human hematopoietic progenitor and stem cells arecharacterized by the presence of a CD34 surface membrane antigen. Thisantigen is used for purification, e.g., on affinity columns which bindCD34. After the bone marrow is harvested, the mononuclear cells areseparated from the other components by means of ficol gradientcentrifugation. This is performed by a semi-automated method using acell separator (e.g., a Baxter Fenwal CS3000+ or Terumo machine). Thelight density cells, composed mostly of mononuclear cells are collectedand the cells are incubated in plastic flasks at 37° C. for 1.5 hours.The adherent cells (monocytes, macrophages and B-Cells) are discarded.The non-adherent cells are then collected and incubated with amonoclonal anti-CD34 antibody (e.g., the murine antibody 9C5) at 4° C.for 30 minutes with gentle rotation. The final concentration for theanti-CD34 antibody is 10 μg/ml. After two washes, paramaoneticmicrospheres (Dyna Beads, supplied by Baxter Immunotherapy Group, SantaAna, Calif.) coated with sheep antimouse IgG (Fc) antibody are added tothe cell suspension at a ratio of 2 cells/bead. After a furtherincubation period of 30 minutes at 4° C., the rosetted cells withmagnetic beads are collected with a magnet. Chymopapain (supplied byBaxter Immunotherapy Group, Santa Ana, Calif.) at a final concentrationof 200 U/ml is added to release the beads from the CD34+ cells.Alternatively, and preferably, an affinity column isolation procedurecan be used which binds to CD34, or to antibodies bound to CD34 (see,the examples below). See, Ho et al. (1995) Stem Cells 13 (suppl. 3):100-105. See also, Brenner (1993) Journal of Hematotherapy 2: 7-17.

In another embodiment, hematopoetic stem cells are isolated from fetalcord blood. Yu et al. (1995) Proc. Natl. Acad. Sci. USA, 92: 699-703describe a preferred method of transducing CD34⁺ cells from human fetalcord blood using retroviral vectors.

B. In vivo Transfection

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic nucleic acids can be administered directly to the organismfor transduction of cells in vivo. Administration is by any of theroutes normally used for introducing a molecule into ultimate contactwith blood or tissue cells. The packaged nucleic acids are administeredin any suitable manner, preferably with pharmaceutically acceptablecarriers. Suitable methods of administering such packaged nucleic acidsare available and well known to those of skill in the art, and, althoughmore than one route can be used to administer a particular composition,a particular route can often provide a more immediate and more effectivereaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the packaged nucleic acidsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, tragacanth, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, fillers, binders, diluents, buffering agents, moisteningagents, preservatives, flavoring agents, dyes, disintegrating agents,and pharmaceutically compatible carriers. Lozenge forms can comprise theactive ingredient in a flavor, usually sucrose and acacia or tragacanth,as well as pastilles comprising the active ingredient in an inert base,such as gelatin and glycerin or sucrose and acacia emulsions, gels, andthe like containing, in addition to the active ingredient, carriersknown in the art.

The packaged nucleic acids, alone or in combination with other suitablecomponents, can be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Suitable formulations for rectal administration include, for example,suppositories, which consist of the packaged nucleic acid with asuppository base. Suitable suppository bases include natural orsynthetic triglycerides or paraffin hydrocarbons. In addition, it isalso possible to use gelatin rectal capsules which consist of acombination of the packaged nucleic acid with a base, including, forexample, liquid triglycerides, polyethylene glycols, and paraffinhydrocarbons.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally, Parenteral administration andintravenous administration are the preferred methods of administration.The formulations of packaged nucleic acid can be presented in unit-doseor multi-dose sealed containers, such as ampules and vials.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by the packaged nucleic acid as described above in thecontext of ex vivo therapy can also be administered intravenously orparenterally as described above.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular vector employed and the condition of thepatient, as well as the body weight or surface area of the patient to betreated. The size of the dose also will be determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, or transduced cell type in aparticular patient.

In determining the effective amount of the vector to be administered inthe treatment or prophylaxis of conditions owing to diminished oraberrant expression of SK or IK channel protein, the physician evaluatescirculating plasma levels of the vector, vector toxicities, progressionof the disease, and the production of anti-vector antibodies. Ingeneral, the dose equivalent of a naked nucleic acid from a vector isfrom about 1 μg to 100 μg for a typical 70 kilogram patient, and dosesof vectors which include a retroviral particle are calculated to yieldan equivalent amount of therapeutic nucleic acid.

For administration, inhibitors and transduced cells of the presentinvention can be administered at a rate determined by the LD-50 of theinhibitor, vector, or transduced cell type, and the side-effects of theinhibitor, vector or cell type at various concentrations, as applied tothe mass and overall health of the patient. Administration can beaccomplished via single or divided doses.

In a preferred embodiment, prior to infusion, blood samples are obtainedand saved for analysis. Between 1×10⁸ and 1×10¹² transduced cells areinfused intravenously over 60-200 minutes. Vital signs and oxygensaturation by pulse oximetry are closely monitored. Blood samples areobtained 5 minutes and 1 hour following infusion and saved forsubsequent analysis. Leukopheresis, transduction and reinfusion can berepeated are repeated every 2 to 3 months. After the first treatment,infusions can be performed on a outpatient basis at the discretion ofthe clinician. If the reinfusion is given as an outpatient, theparticipant is monitored for at least 4, and preferably 8 hoursfollowing the therapy.

Transduced cells are prepared for reinfusion according to establishedmethods. See, Abrahamsen et al. (1991) J. Clin. Apheresis, 6: 48-53;Carter et al. (1988) J. Clin. Arpheresis, 4:113-117; Aebersold et al.(1988) J. Immunol. Meth., 112: 1-7; Muul et al. (1987) J. ImmunolMethods, 101:171-181 and Carter et al. (1987) Transfusion 27: 362-365.After a period of about 2-4 weeks in culture, the cells should numberbetween 1×10⁸ and 1×10¹². In this regard, the growth characteristics ofcells vary from patient to patient and from cell type to cell type.About 72 hours prior to reinfusion of the transduced cells, an aliquotis taken for analysis of phenotype, and percentage of cells expressingthe therapeutic agent.

Although the present invention has been described in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious that certain changes and modifications may be practicedwithin the scope of the appended claims.

EXAMPLE 1

Example 1 describes the isolation and sequencing of clones encodingsmall and intermediate conductance, calcium-dependent potassiumchannels.

A. Small conductance potassium channels, with the exception of the minkprotein (Takumi et al., Science, 242:1042-1045 (1988), share a commonstructural motif within the pore region including the sequence whichdictates the characteristic selectivity sequence for monovalent cations(Heginbotham et al., Biophys. J., 66:1061-1067 (1994)).

A BLAST search of the EST database using the query sequenceFXSIPXXXWWAXVTMTTVGYGDMXP (SEQ ID NO:45), allowing for mismatches,retrieved known potassium channel sequences and Genbank #M62043.Oligonucleotides corresponding to nucleotides 6-36 (sense) and 258-287(antisense) of #M62043 were synthesized (Genosys, The Woodlands, Tex.),radiolabeled using polynucleotide kinase (BRL) and ³²P-ATP (NEN), andused to screen ˜10⁵ recombinant phage from the human hippocampal cDNAlibrary (40% formamide; 1 M NaCl, 1% SDS, 37° C.; washed at 1×SSC, 50°C.). Double positively hybridizing phage were purified by rescreening atreduced densities. cDNA inserts were subcloned into M13 and thenucleotide sequences determined using the dideoxy chain terminationmethod and T7 DNA polymerase (Sequenase, UBI). A fragment of this clonecontaining the pore domain (amino acids 325-522) was radiolabeled usingrandom primers (Boehringer) and used to screen a rat brain cDNA library(30% formamide, 1 M NaCl, 1% SDS, 37° C.; washed at 2×SSC, 50° C.).Positively hybridizing phage were purified and the nucleotide sequencesof the inserts determined. Computer analyses were performed using theGCG software suite (Genetics Computer Group; version 8.1).

In addition to known potassium channels, one of the detected sequencesfrom human hippocampus suggested it may contain the consensus motif, butincluded several ambiguities (Genbank #M62043). Based upon thissequence, oligonucleotides were synthesized having the sequencerepresented by nucleotides 6 to 36 of the sense strand; and nucleotides258 to 287 of the antisense strand. The oligonucleotides were used toprobe a human hippocampal cDNA library.

A full length coding sequence, hSK1 (SEQ ID NO:13), was isolated andanalyzed for open reading frames, Kozak consensus sequences, potentialtransmembrane domains, and predicted protein structure. A fragmentcontaining the putative pore region was radiolabelled by random primingand subsequently used to probe a rat brain cDNA library using ahybridization solution of 40% formamide, 1 M NaCl, 1% SDS, and 100 μg/mlyeast RNA, at 37° C. and washed using 0.5×SSC at 55° C. Two clonescontaining different full length coding sequences were isolated andanalyzed: rSK2 (SEQ ID NO:15), and rSK3 (SEQ ID NO:16). In addition, apartial clone was identified representing the rat homolog of hSK1 (rSK1(SEQ ID NO:14)).

The sequences predict proteins of 561 amino acids for hSK1 (SEQ IDNO:1), 580 amino acids for rSK2 (SEQ ID NO:2), and 553 amino acids forrSK3 (SEQ ID NO:3) which contain no stretches of homology (i.e., nosignal above background under low stringency conditions) with othercloned potassium channels apart from a 12 amino acid sequence in theputative pore region. Hydrophobicity analysis predicts six transmembranesegments with the N- and C-termini residing inside the cell. Thesequences are highly conserved across their transmembrane cores (80-90%identity), but diverge in sequence and length within their N- andC-terminal domains (Table 1).

TABLE I rSK2 .......... ........MS SCRYNGGVMR PLSNLSSSRR NLHEMDSEAQ rSK3.......... ........MS SCKYSGGVMK PLSRLSASRR NLIEAEPEGQ rSK1 .................... .......... .......... .......... hSK1 MPGPRAACSE PNPCTQVVMNSHSYNGSVGR P...LGSGPG ALGRDPPDPE rSK2 PLQPPASVVG GGGGASSPSA AAAASSSAPEIVVSKPEHNN SNNLALYGTG rSK3 PLQLF..... .......... ...SPSNPPE IIISSREDNHAHQTLLHHPN rSK1 .......... .......... .......... .......... ..........hSK1 AGHPPQPPHS PGLQVVVAKS EPARPSPGSP RGQPQDQDDD EDDEEDEAGR rSK2GGGSTGGGGG GGGGGGGSGH GSSSGTKSSK KKNQNIGYKL GHRRALFEKR rSK3 ATHNHQHAGTTAGSTTFP.. ......KANK RKNQNIGYKL GHRRALFEKR rSK1 .......... ...................S GKPPTVSHRL GHRRALFEKR hSK1 QR........ .......... ........ASGKPSNVGHRL GHRRALFEKR rSK2 KRLSDYALIF GMFGIVVMVI ETELSWGAYD KASLYSLALKCLISLSTIIL rSK3 KRLSDYALIF GMFGIVVMVI ETELSWGLYS KDSMFSLALK CLISLSTIILrSK1 KRLSDYALIF GMFGIVVMVT ETELSWGVYT KESLCSFALK CLISLSTVIL hSK1KRLSDYALIF GMFGIVVMVT ETELSWGVYT KESLYSFALK CLISLSTAIL rSK2 LGLIIVYHAREIQLFMVDNG ADDWRIAMTY ERIFFICLEI LVCAIHPIPG rSK3 LGLIIAYHTR EVQLFVIDNGADDWRIAMTY ERILYISLEM LVCAIHPIPG rSK1 LGLVILYHAR EIQLFLVDNG ADDWRIAMTWERVSLISLEL AVCAIHPVPG hSK1 LGLVVLYHAR EIQLFHVDNG ADDWRIAMTC ERVFLISLELAVCAIHPVPG rSK2 NYTFTWTARL AFSYAPSTTT ADVDIILSIP MFLRLYLIAR VMLLHSKLFTrSK3 EYKEEWTARL AFSYTPSRAE ADVDIILSIP MFLRLYLIAR VMLLHSKLFT rSK1HYRFTWTARL AFSLVPSAAE ADVDVLLSIP MFLRLYLLAR VMLLHSRIFT hSK1 HYRFTWTARLAFTYAPSVAE ADVDVLLSIP MFLRLYLLGR VMLLHSKIFT rSK2 DASSRSIGAL NKINFNTRFVMKTLMTICPG TVLLVFSISL WIIAAWTVRA rSK3 DASSRSIGAL NKINFNTRFV MKTLMTICPGTVLLMFSISL HIIAAWTVRV rSK1 DASSRSIGAL NRVTFNTRFV TKTLMTICPG TVLLVFSISSWIVAAHTVRV hSK1 DASSRSIGAL NKITFNTRFV MKTLMTICPG TVLLVFSISS WIIAAWTVRVrSK2 CERYHDQQDV TSNFLGAMWL ISITFLSIGY GDMVPNTYCG KGVCLLTGIM rSK3CERYHDQQDV TSNFLGAMWL ISITFLSIGY GDMVPNTYCG KGVCLLTGIM rSK1 CERYHDKQEVTSNFLGAMWL ISITFLSIGY GDMVPNTYCG KGVCLLTGIM hSK1 CERYHDKQEV TSNFLGAMWLISITFLSIGY GDMVPNTYCG KGVCLLTGIM rSK2 GAGCTALVVA VVARKLELTK AEKHVHNFMMDTQLTKRVKN AAANVLRETW rSK3 GAGCTALVVA VVARKLELTK AEKHVHNFMM DTQLTKRIKNAAANVLRETW rSK1 GAGCTALVVA VVARKLELTK AEKHVHNFMM DTQLTKRVKN AAANVLRETWhSK1 GAGCTALVVA VVARKLELTK AEKHVHNFMM DTQLTKRVKN AAANVLRETW rSK2LIYKNTKLVK KIDHAKVRKH QRKFLQAIHQ ...LRSVKME QRKLNDQANT rSK3 LIYKHTKLLKKIDHAKVRKH QRKFLQAIHQ ...LRGVKME QRKLSDQANT rSK1 LIYKHTRLVK KPDQSRVRKHQRKFLQAIHQ AQKLRTVKIE QGKVNDQANT hSK1 LIYKHTRLVK KPDQARVRKH QRKFLQAIHQAQKLRSVKIE QGKLNDQANT rSK2 LVDLAKTQNI MYDMISDLNE RSEDFEXRIV TLETKLETLIGSIHALPGLI rSK3 LVDLSKMQNV MYDLITELND RSEDLEKQIG SLESKLEHLT ASFNSLPPLIrSK1 LADLAKAQSI AYEVVSELQA QQEELEARLA ALESRLDVLG ASLQALPSLI hSK1LTDLAKTQTV MYDLVSELHA QHEELEARLA TLESRLDALG ASLQALPGLI rSK2 SQTI....RQQQRDFIETQM ENYDKHVTYN AERSRSSSRR RRSSSTAPPT rSK3 ADTLRQQQQQ LLTAFVEARGISVAVG.... .......... ...TSHAPPS rSK1 AQAICPLPPP W...PGPSHL TTAAQSPQSHWLPTTASDCG *......... hSK1 AQAIRPPPPP LPPRPGPGPQ DQAARSSPCR WTPVAPSDCG*......... rSK2 SSESS..... ........ rSK3 DSPIGISSTS FPEFLIF* rSK1.......... ........ hSK1 .......... ........The fourth predicted membrane spanning domain contains 3 positivelycharged residues that do not occupy every third position as involtage-dependent potassium channels (Durell et al., Biophys. J.,62:238-250 (1992)), but are separated by 6 and 7 residues. There aremultiple consensus targets for phosphorylation by a variety of proteinkinases. Some of these sites are found in all clones. However, eachclone contains potential phosphorylation sites not conserved among allmembers. There are no conserved N-linked glycosylation sites (NXXS/T)(SEQ ID NO:46) in predicted extracellular domains, and no consensusnucleotide or calcium binding domains (E-F hands).

Northern blots of rat brain and skeletal muscle showed that rSK3transcripts from these tissues encoded proteins that were N-terminallyextended relative to the rSK3 clone SEQ ID NO:16. The nucleic acidencoding the rSK3 N-terminal extension was cloned and sequenced, and thecDNA encoding N-terminal extended rSK3 is represented by SEQ ID NO:44.In addition, endogenous rSK3 was shown to have a nucleotide sequencethat encodes a protein having a C-terminus with the last 5 amino acidsof SEQ ID NO:3 replaced by the last 9 amino acids of SEQ ID NO:43.Similarly, hSK3 was shown to have an N-terminal extension, and the cDNAencoding this N-terminal extension is represented by SEQ ID NO:48.

B. To isolate intermediate conductance calcium activated K⁺ proteins,one can use PCR under standard conditions. Suitable primers are SEQ IDNOS:34 and 35 which yield a probe of about 270 bases and SEQ ID NOS:36and 37 which yield a probe of about 165 bases. These primers can be usedto amplify plasmid DNA comprising cloned hIK1 or on reverse transcribedRNA from a tissue which expresses hIK1, such as a cDNA library frompancreas. The PCR reaction will yield DNA fragments of the specifiedsize which contain sequences specific to hIK1 and related genes. TheseDNA fragments are subsequently labeled for use as hybridization probesby standard random-priming protocols. The labeled probes are then usedto screen libraries at high stringency to isolate only hIK1 sequences,or at moderately low stringency (30-40% formamide, 37° C. hyb/1×SSC, 55°C. wash) to isolate putatively related sequences. Alternatively, one canamplify the intact hIK1 gene from a pancreas cDNA library using PCRprimer pair SEQ ID NOS:38 and 39 or 40 and 41.

EXAMPLE 2

Example 2 describes in situ hybridization of rat brain sections usingsequences distinct for each of the rat SK channel clones, anddetermination of transcript sizes from various peripheral tissues.

Care and handling of adult female Sprague-Dawley rats were in accordancewith the highest standards of institutional guidelines. Rats were deeplyanesthetized with pentobarbital and perfused transcardially withice-cold saline, followed by ice-cold 4% paraformaldehyde in 0.1 Msodium borate (pH 9.5). The brains were removed quickly and post-fixedovernight at 4° C. in 4% paraformaldehyde in borate buffer (pH 9.5)containing 10% sucrose. Cryostat microtome sections (25 mm) were mountedonto gelatin- and poly-L-lysine-coated glass slides and incubated for 15min in 4% paraformaldehyde in 0.1 M PBS, washed twice in 0.1 M PBS, andtreated for 30 min at 37° C. in 10 mg/ml proteinase K in 100 mM Tris, 50mM EDTA (pH 8), followed by 0.0025% acetic anhydride in 0.1 Mtriethanolamine at room temperature. The sections were then washed in2×SSC, dehydrated in increasing concentrations of ethanol, andvacuum-dried at room temperature.

Templates for probe synthesis represented C-terminal and 3′ untranslatedsequences unique to each of the clones, and were subcloned into pKS.Using linearized template DNA, ³⁵S -labeled antisense cRNA probe heatedto 65° C. for 5 min and diluted to 10⁷ cpm/ml in hybridization buffer;66% formamide, 260 mM NaCl, 1.3×Denhardt solution, (13 mM Tris, pH 8.0,1.3 mM EDTA, 13% dextran sulfate). Sections in hybridization mixturewere covered with siliconized glass coverslips and sealed using DPXmountant. After incubating at 58° C. for 20 hr, the slides were soakedin 4×SSC to remove coverslips, then rinsed in 4×SSC (4 times, 5 mineach) prior to ribonuclease A treatment (20 mg/ml for 30 min at 37° C.).The slides were then rinsed in decreasing concentrations of SSCcontaining 1 mM DTT to a final stringency of 0.1×SSC, 1 mM DTT for 30min at 65° C. After dehydrating the sections in increasingconcentrations of ethanol, they were vacuum-dried and exposed to DuPontCronex-4 X-ray film for 7 days. The film was scanned by a MicrotekScanMaker 1850S at 728 pixel/cm resolution and the images analyzed usingImage v1.55 software (NIH) and Photoshop (Adobe).

The results iddicate that mRNAs to the rat sequences are broadlydistributed throughout the CNS, in characteristic but overlappingpatterns. rSK1 is expressed in the hippocampus and the dentate gyrus,the granular layer of the cerebellum, and the anterior olfactorynucleus. rSK1 mRNA was also detected in the subiculum, the olfactorytubercle, and the neocortex. rSK2 mRNA is the most widely expressed,with highest expression in the hippocampus and lower levels in thedentate gyrus, the olfactory bulb and the anterior olfactory nucleus.rSK2 mRNA was also detected in the granular layer of the cerebellum, thereticular nucleus of the thalamus, and the pontine nucleus. The patternof in situ hybridization for rSK2 mRNA is coincident with the pattern ofradiolabeled apamin binding in rat brain (Gelhart, Neuroscience,52:191-205 (1993)). rSK3 mRNA was detected in the olfactory tubercle andolfactory bulb, throughout the thalamus, the lateral septum, the ventraltegmental area, and the substantia nigra pars compacta. Moderate levelswere detected throughout the hypothalamus, the caudate putamen, and thenucleus accumbens.

The same distinct sequences for rSK1 and rSK2 were used to probeNorthern blots prepared with mRNA isolated from total brain and severalperipheral tissues. Total RNA was extracted (Chirgwin et al., Biochem.,18:5294-5300 (1979)) from rat brain, adrenal gland, thymus, spleen,skeletal muscle, heart, kidney, liver, and lung of 3 week oldSprague-Dawley rats. Poly (A)⁺ mRNA was purified by oligo d(T) cellulosechromatography (Collaborative Research), and 3 μg from each tissue wasprepared as a Northern blot by electrophoresis through a 1%agarose-formaldehyde gel and transfer to Genescreen (NEN) nylonmembranes. Antisense riboprobes of the same sequence as used for in situhybridization were synthesized from linearized DNA templates using³²P-UTP (NEN). Blots were hybridized in 50% formamide, 5% SDS, 400 mMNaPO₄, 1 mM EDTA at 60° C. for 12 hours, followed by washes in 0.05×SSCat 65° C., and visualized using a Phosphorimager 445 SI (MolecularDynamics) after 15 hours.

rSK1 mRNA was detected in rat brain and heart, while rSK2 mRNA wasdetected in brain and adrenal gland. The results show that rSK1mRNAs ofdifferent sizes are present in brain (3.2 kb) and heart (4.4 kb). rSK2mRNA was detected in brain and adrenal gland as two bands of 2.2 and 2.4kb. Neither rSK1 nor rSK2 mRNA was detected from lung, liver, kidney,thymus, spleen, or skeletal muscle.

EXAMPLE 3

Example 3 describes in vitro expression of SK and IK channel proteins.

3A. Example 3A describes in vitro expression of rSK2 and hSK1 mRNAs inXenopus oocytes and measurements of electrical conductance.

In vitro mRNA synthesis and oocyte injections were performed asdescribed in Adelman et al., Neuron, 9:209-216 (1992). Xenopus care andhandling were in accordance with the highest standards of institutionalguidelines. Frogs underwent no more than two surgeries, separated by atleast three weeks, and surgeries were performed using well establishedtechniques. Frogs were anesthetized with an aerated solution of3-aminobenzoic acid ethyl ester.

Oocytes were studied 2-5 days after injection with 2 ng of mRNA. Wholecell currents were measured after mRNA injection using a two electrodevoltage clamp with a CA-1 amplifier interfaced to a Macintosh Quadra 650computer. Data were simultaneously acquired through Pulse (Heka,Germany) at 500 Hz and Chart (AD Instruments, Australia) at 10 Hz.During recording, oocytes were continuously superfused with ND-96solution containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl_(2,) 1 mMMgCl_(2,) 5 mM HEPES (pH 7.5 with NaOH) at room temperature. To minimizeCl⁻ currents, some oocytes were soaked and studied in Cl⁻-free ND96solution (96 mM Nagluconate, 2 mM Kgluconate, 2.7 mM Cagluconate₂, 1 mMMggluconate_(2,) 5 mM HEPES, pH 7.5 with NaOH). Voltage protocols from aholding potential of −80 mV failed to evoke currents different fromcontrol oocytes.

Because the expression pattern of rSK2 is similar to that of mGluR1a, ametabotropic glutamate receptor (Houamed et al., Science, 252:1318-1321(1991); Masu et al., Nature, 349:760-765 (1991)), mGluR1a mRNA wasinjected with or without the SK mRNAS. Addition of glutamate (1 mM) tothe bath comprising the oocyte injected with mGluR1a mRNA alone evoked atransient inward current due to activation of endogenouscalcium-activated chloride channels following the release ofintracellular calcium (Houamed et al., Science, 252:1318-1321 (1991);Masu et al., Nature, 349:760-765 (1991)). Similar results were obtainedin six other oocytes injected with mGluR1a. Voltage ramps from −120 to60 mV applied near the peak of the inward response evoked an outwardlyrectifying current that reversed at −25 mv, near the Cl⁻ reversalpotential. Addition of glutamate (1 mM) to oocytes coinjected withmGluR1a and rSK2 mRNA evoked the transient calcium-activated chloridecurrent observed with mGluR1a injected oocytes, followed by a largetransient outward current. Similar results were obtained in 14 otheroocytes coinjected with mGluR1a and rSK2. Voltage ramps from −120 to 60mV applied near the peak of the outward response evoked a large inwardlyrectifying current that reversed near −95 mV, close to the K⁺ reversalpotential. This result was obtained with each of the cloned subunits andsuggested that the cloned sequences encode potassium channels.

Following establishment of the 2-electrode voltage clamp, the oocyte wasimpaled with a third electrode containing 200 mM EGTA, pH adjusted to7.2 with KOH. The input resistance was monitored during impalement toinsure oocyte viability. At the indicated time, 50 nl of the EGTAsolution was injected into the oocyte. Assuming an oocyte volume of 1μl, the predicted final concentration of EGTA was 10 mM. Intracellularinjection of EGTA abolished both current responses evoked by subsequentapplication of glutamate indicating that both components arecalcium-activated. Similar results were obtained in 3 other oocytescoinjected with mGluR1a and rSK2. Current-voltage relation of oocytesinjected with rSK2 mRNA in Cl⁻-free external solution containing 2, 6 or20 mM K⁺. The current was activated by injection of CaCl₂ to a finalconcentration of ˜1 mM (Adelman et al., Neuron, 9:209-216 (1992)).Background current was determined by application of 100 nM apamin. Theapamin-insensitive background current did not vary with external K⁺.

Two days after injection, the oocytes were soaked for >24 hours in Cl⁻free ND96 solution to minimize Cl⁻ currents. In the 2-electroderecording mode, the channel was activated by injection of 5 nl of 200 mMCaCl₂ through a third electrode resulting in a final intracellularconcentration of ˜1 mM Ca²⁺. This procedure resulted in a longer lastingactivation of the K⁺ current than that activated by glutamate in oocytescoinjected with mGluR1a and rSK2. In these oocytes, the reversalpotential was determined relative to background current in 100 nMapamin. The mean reversal potential ±S.D. plotted versus [K⁺]_(o) yieldsa slope of 55.4 mV/decade change in [K⁺]_(o) and a y-intercept of −110mV at 1 mM [K⁺]_(o).

Macroscopic currents were also recorded from excised patches. Currentswere elicited by 2.5 second voltage ramps from −100 to 100 mV in anexcised inside-out patch from an oocyte expressing rSK2. Without bathapplied calcium, currents were not different from control oocytes.Oocytes were injected as described for two-electrode voltage damprecordings.

Two to nine days after injection, inside-out macropatches were excisedinto a bath solution containing 116 mM Kgluconate, 4 mM KCl, 10 mM HEPES(pH 7.25, adjusted with KOH) supplemented with CaCl₂ and/or EGTA. Toobtain nominally Ca-free solution, 1 mM EGTA was added. Alternatively,CaCl₂ was added to the bath solution to give free calcium concentrationsof 1-10 μM. In this case, the proportion of calcium binding to gluconatewas determined by a computer program (CaBuf) assuming a stabilityconstant for Ca²⁺ gluconate of 15.9 M⁺¹ (Dawson et al., Data forBiochemical Research (Oxford University Press, New York, (1969)). Toobtain Ca²⁺ concentrations below 1 μM, 5 mM EGTA was added to the bathsolution and CaCl₂ was added as calculated using the CaBuf program andpublished stability constants (Fabiato et al., J. Physiol., 75:463-505(1979)). For experiments in which Mg²⁺ was added to the bath solution,MgCl₂ was added to the total concentrations stated in the text. Underthese conditions, binding of Mg²⁺ to gluconate is negligible (stabilityconstant 1.7 M⁻¹).

Electrodes were pulled from thin-walled, filamented borosilicate glass(World Precision Instruments) and filled with 116 mM Kgluconate, 4 mMKCl, 10 mM HEPES (pH 7.25). Electrode resistance was typically 2-5 MΩ.Membrane patches were voltage clamped using an Axopatch 200A amplifier(Axon Instruments). The data were low-pass Bessel filtered at 2 kH andacquired using Pulse software (HEKA Electronik). Analysis was performedusing Pulse, Kaleidograph (Abelbeck), or IGOR (Wavemetrics) software.All experiments were performed at room temperature from a holdingpotential of −80 mV. 2.5 second voltage ramps from −100 to 100 mV wereacquired at a sampling frequency of 500 Hz. Alternatively,current-voltage relationships were obtained from the mean current during500 ms commands to voltages between −100 and 100 mV in 20 mV increments,sampled at 5 kHz.

Addition of 5 μM Ca²⁺ to the intracellular (bath) solution evoked asubstantial current. Voltage ramps in symmetrical 120 mM K³⁰ and in theabsence of internal Mg²⁺ revealed a current-voltage relationship withslight inward rectification. Voltage steps between −100 and 100 mV, froma holding potential of −80 mV, evoked time-independent currents. Thederived I-V relationship reflects the inward rectification apparent fromvoltage ramps. The current was evoked by voltage steps from aninside-out macropatch excised from an oocyte expressing rSK2. With 5 μMCa²⁺ in the bath, the membrane was stepped from a holding potential of−80 mV to test potentials between −100 and 100 mV and then repolarizedto −50 mV. Currents activated instantaneously and showed no inactivationduring the 500 ms test pulses. Similar results were obtained for hSK1,except that the inward rectification was not as pronounced. Theseresults identify this new family as calcium-activated potassiumchannels.

3B. Example 3B describes the electrophysiology of the hIK1 channel. AllhIK1 channel subunits were subcloned into the oocyte expression vectorpBF (unpublished, graciously provided by Dr. B. Fakler) which provides5′ and 3′ untranslated regions from the Xenopus β-globin gene flanking apolylinker containing multiple restriction sites. In vitro mRNAs weregenerated using SP6 polymerase (GibcoBRL); following synthesis, mRNAswere evaluated spectrophotometrically and by ethidium bromide stainingafter agarose gel electrophoresis.

As described above, Xenopus care and handling were in accordance withthe highest standards of institutional guidelines. Frogs underwent nomore than two surgeries, separated by at least three weeks, and Allsurgeries were performed using well established techniques. Frogs wereanesthetized with an aerated solution of 3-aminobenzoic acid ethylester. Oocytes were studied 2-14 days after injection with 0.5-5 ng ofmRNA.

Inside-out macropatches were excised into an intracellular solutioncontaining 116 mM K-gluconate, 4 mM KCl, 10 mM HEPES (pH 7.2, adiustedwith KOH) supplemented with CaCl₂ to give free calcium concentration of5 μM; the proportion of calcium binding to gluconate was determined by acomputer program (CaBuf) assuming a stability constant for Ca²⁺gluconate of 15.9 M⁻¹ (Dawson et al., 1969). To obtain Ca²⁺concentrations below 1 μM, 1 mM EGTA was added to the bath solution andCaCl₂ was added as calculated using the CaBuf program and publishedstability constants (Fabiato and Fabiato, 1979). Electrodes were pulledfrom thin-walled, filamented borosilicate glass (World PrecisionInstruments) and filled with 116 mM K-gluconate, 4 mM KCl, 10 mM HEPES(pH 7.2). Electrode resistance was typically 2-5 MΩ. For outside-outmacropatches, the solutions were reversed. Membrane patches were voltageclamped using an Axopatch 200A amplifier (Axon Instruments). The datawere low-pass Bessel filtered at 1 kH and acquired using Pulse software(HEKA Electronik). Analysis was performed using Pulse, Kaleidagraph(Abelbeck), or IGOR (Wavemetrics) software. Unless otherwise stated allexperiments were performed at room temperature from a holding potentialof 0 mV. 2.5 second voltage ramps from −100 to either 60 or 100 mV wereacquired at a sampling frequency of 500 Hz. Values were expressed asmean ±SD. Statistical differences were determined using an unpairedt-test; p values <0.05 were considered significant.

For single channel recordings, oocytes were bathed in 116 mM Kgluconate,4 mM KCl, 10 mM HEPES, 5 mM EGTA, pH 7.2 adjusted with CaCl₂ to yieldthe reported concentration of free Ca²⁺. All recordings were performedin the inside-out patch configuration using thick-walled quartzelectrodes (13-15 MΩ) containing 116 mM Kgluconate, 4 mM KCl, 10 mMHEPES, pH 7.2. Membrane patches were voltage-clamped with an Axopatch200 amplifier (Axon Instruments). Continuous recordings were low-passBessel filtered at 1 kHz, acquired at 10 kHz using Pulse software (HekaElectronik) and stored directly on a Macintosh Quadra 650. Singlechannel recordings were analyzed with MacTac (SKALAR Instruments) usingthe “50% threshold” technique to estimate event amplitudes and duration,and each transition was visually inspected before being accepted.Amplitude histograms were constructed using MacTacfit (SKALARInstruments). Only events tasting at least 1 ms were included, andamplitude histograms were fitted by single Gaussian distributions. Allexperiments were performed at room temperature.

The expression of the hIK1 in Xenopus oocytes was readily detectable.Voltage ramp commands delivered to inside-out patches excised into 5 μMCa²⁺ evoked robust, inwardly rectifying macroscopic current responses,not present in patches from uninjected oocytes (not shown) or inside-outpatches bathed in Ca²⁺-free media. Voltage step commands evoked largetime-independent currents only when Ca²⁺ was included in the (bath)internal solution. Altering the external K⁺ concentration (substitutedby Na) shifted the reversal potential in accord with the Nernstprediction for a K⁺-selective conductance (57 mV/10-fold change in K⁺).Similar to SK2 channels, currents evoked by voltage ramp commands weredependent upon the concentration of Ca²⁺ applied to the internal face ofthe membrane.

EXAMPLE 4

Example 4 describes the calcium sensitivity of rSK2 and hSK1 channels.

Using inside-out micropatches as described above, rSK2 currents evokedby voltage ramps were shown to dependent upon the concentration ofcalcium in the internal (bath) solution. The slope conductance at thereversal potential was plotted as a function of calcium concentrationand the data points fit with the Hill equation. From 8 patches, theaverage K_(d) for calcium was 0.63±0.23 μM. The steep dependence uponcalcium seen from the plot is reflected by a Hill coefficient of4.81±1.46, suggesting that at least two calcium ions are involved inchannel gating. Similar experiments performned with hSK1 yielded a K_(d)of 0.70±0.06 μM and a Hill coefficient of 3.90±0.45.

To compare hIK1 and SK2, normalized current was plotted as a function ofCa²⁺ concentration, and the data points fitted with the Hill equation.Both channels showed the same K_(0.5) (concentration for half-maximalactivation, 0.32±0.03 μM (n=7) for hIK1 and 0.31±0.05 μM (n=4) for SK2;p=0.68), but differed in the steepness of the Ca²⁺-dependence; SK2 had aHill coefficient of 3.5±0.4 (n=4), while hIK1 had a Hill coefficient of1.7±0.3 (n=7, p<0.001). These results demonstrate that hIK1 is also acalcium-activated potassium channel.

EXAMPLE 5

Example 5 describes the magnesium induced inward rectification for therSK2 channel.

The inward rectification for rSK2, described above, was observed in theabsence of internal cations other than potassium and calcium (5 μM).Native SK channels exhibit inward rectification induced by internal Mg²⁺ions (Lancaster et al., J. Neurosci., 11:23-30 (1991)). In thehippocampus, SK channels exhibit significant inward rectification in thepresence of internal Mg²⁺ (Id.). Currents were elicited from aninside-out macropatch excised from an oocyte expressing rSK2 in thepresence of the varying concentrations of internal Mg²⁺ and 10 μM Ca²⁺.When different concentrations of Mg²⁺(0.1-3 mM) were added to thesolution bathing inside-out patches, outward currents were significantlyreduced.

The concentration- and voltage-dependence of Mg²⁺ induced inwardrectification was examined. A slight decrease of the inward current withincreasing Mg²⁺ was observed. Therefore, the ratio of the outwardcurrent at potentials between 20 and 100 mV to the inward current at−100 mV was plotted as a function of the different concentrations ofinternal Mg²⁺. From multiple experiments, the data points obtained atdifferent Mg²⁺ concentrations and voltages were fit with the Hillequation, yielding an average Hill coefficient, of 0.94±0.27 (n=24).Subsequently, the Hill coefficient was fixed at 1, and the mean K_(d)was plotted as a function of the test potential. The K_(d) decreasedwith increasing voltages suggesting that Mg²⁺ block wasvoltage-dependent. K_(d) for Mg²⁺ was obtained from 5 patches as shownin panel B at 20, 40, 60, 80 and 100 mV. Values at each potential wereaveraged, plotted as a function of voltage and fit with the Woodhullequation, K_(d)(0 mV)exp(δzFE/RT) where the K_(d)(0 mV)=6 mM, δ is thefraction of the electric field sensed by the Mg²⁺ ion, 0.30, z is thevalence, 2, and F, E, R, and T have their usual meanings (Woodhull, J.Gen. Physiol., 61:687-708 (1973)). Applying the Woodhull equationsuggested that the Mg²⁺ ion senses approximately 0.30 of the membraneelectric field.

EXAMPLE 6

Example 6 describes single channel recordings from oocytes.

6A. Example 6A describes single channels were examined using inside-outpatches excised from oocytes expressing rSK2. Addition of calcium atsubmicromolar concentrations induced channel activity not seen incontrols. A representative patch showed that 0.2 μM calcium applied tothe bath solution induced openings to a single amplitude. Channelactivity increased as the calcium concentration was raised, such that in0.6 μM calcium unitary openings could no longer be resolved. Uponwashout of calcium, channel activity disappeared. Channel activity inthe presence of 0.4 μM calcium was recorded at several voltages. Similarto macroscopic ramp recordings, channel open probability was notobviously dependent upon voltage.

Unitary openings measured at several voltages were used to construct asingle channel I-V relationship. Solutions used were the same as formacropatch recordings (Example 5). Electrodes were pulled from Corning7052 glass (Garner) and had resistances of 9-13 MΩ. Data were filteredat 1 kHz (Bessel), acquired at 10 kHz using Pulse (HEKA Electronik) andstored directly on a Macintosh Quadra 650. Single channels were analyzedusing MacTac (SKALAR Instruments). The “50% threshold” technique wasused to estimate event amplitudes. The threshold was adjusted for eachopening and each transition was inspected visually before beingaccepted. Amplitude histograms were constructed using MacTacfit (SKALARInstruments) and best fit by a single Gaussian distribution. Channelopen probability was estimated as NP(o), the product of the openprobability multiplied by the number of channels. NP(o) was calculatedas the sum of the (dwell time×level number) divided by the total time. Nwas estimated as the number of simultaneously open channels at 0.4 μMcalcium. Linear regression analysis on three patches from an oocyteexpressing either rSK2 or hSK1 yielded a mean single channel conductanceof 9.9±0.9 pS and 9.2±0.3 pS, respectively.

6B. Example 6B describes the single channel conductance of hIK1. Themethodology is described above in Example 3B. Stationary recordings frominside-out patches excised into a bathing solution containing 0.2-1.0 μMfree calcium showed short-duration openings not seen in the absence ofcalcium. Representative traces were recorded at −60 mV. The degree ofchannel activity depended upon the concentration of internal calcium.Reducing intracellular calcium reduced channel activity, and removinginternal calcium abolished channel activity, which returned afterreapplication of Ca²⁺. Sustained channel activity was seen at membranevoltages ranging from −100 mV to +100 mV and open probability was notobviously voltage-dependent. For select patchs, the amplitudes ofopenings were measured, assembled into histograms, and fit by Gaussiandistributions. The resulting mean amplitudes were used to construct thecurrent-voltage relationship. The single channel current-voltagerelationship shows inward rectification similar to the macroscopiccurrent-voltage relationship. For this patch, linear regression analysisof the inward current-voltage relationship yielded a single channelconductance of 35 pS; results from four patches gave a unit conductanceof 38±4 pS. Measurements of the outward conductance were more variable,ranging from 5 to 12 pS.

EXAMPLE 7

Example 7 describes the pharmacology of the novel rat and humanpotassium channels.

7A. Macroscopic rSK2 currents were recorded in 5 μM Ca²⁺ from inside-outmacropatches with either 0 or 60 μM apamin or 0 or 2 pM d-tubocurare inthe patch pipette described in Example 3. The functional characteristicsof the cloned channels are reminiscent of the SK class ofcalcium-activated potassium channels described in neurons (Lancaster andAdams, J. Neurophysiol, 55:1268-1282 (1986); Lancaster et al., J.Neurosci., 11:23-30 (1991); Sah et al., J. Neurophysiol., 68:1834-1841(1992)), skeletal muscle (Blatz and Magleby, Nature, 323:718-720(1986)), adrenal chromaffin cells (Park, J. Physiol., 481:555-570((1994); Artalejo et al., Pflugers Archiv., 423:97-103 (1993)), andT-lymphocytes (Grissmer et al., J. Gen. Physiol., 99:63-84 (1992)).Native SK channels present a distinct pharmacology. They are not blockedby the scorpion peptide, charybdotoxin (CTX), a potent blocker of BKpotassium channels (Miller et al., Nature, 313:316-318 (1985)). However,many but not all SK channels are blocked by the bee venom toxin, apamin,and the plant alkyloid, d-tubocurare (dTC; Zhang and McBain, J.Physiol., 488:661-672 (1995), Park, J. Physiol., 481:555-570 (1994); Dunet al., J. Physiol., 375:499-514 (1986)). Application of 500 nM CTX didnot block rSK2 or hSK1, but abolished the activity of hSlo BK currents.rSK2 currents were potently blocked by picomolar concentrations ofapamin with a K_(d) of 63 pM. In contrast, application of 100 nM apamindid not affect hSK-1 currents (n=8). dTC also blocked rSK2 currents witha K_(d) of 2.4 μM, while hSK1 was approximately 30-fold less sensitive,with a K_(d) of 76.2 μM.

7B. For the pharmacology tests of hIK1, Clotrimazole was from Sigma,ketoconazole and iberiotoxin were from RPI, apamin was from Calbiochem,charybdotoxin was the generous gift of Dr. Chris Miller. The functionalcharacteristics of hIK1 are remeniscent of intermediate conductancecalcium-activated K⁺ channels described from red blood cells (the Gardoschannel; Gardos, 1958) and other tissues. Native IK channels present adistinguishing pharmacology, being blocked by charybdotoxin (CTX) but,different from large conductance voltage- and Ca²⁺-activated K⁺ channels(BK channels), are not blocked by iberiotoxin. Also, IK channels are notsensitive to the bee venom peptide toxin apamin, a blocker of certainnative and cloned SK channels. In addition, some IK channels, notablythe Gardos channel, are sensitive to several imidazole derivatives suchas clotrimazole, but are not sensitive to others such as ketoconazole.hIK1 currents were potently blocked by CTX, with a K_(i), of 2.5 nM(n=4), while 50 nM IBX blocked only 15±3%. Human IK1 was sensitive toclotrimazole with a Ki of 24.8 nM, but was only 24±6% blocked by 10 μMketoconazole. 100 nM apamin reduced hIK1 currents by only 12±5%.

All publications and patents mentioned in this specification are hereinincorporated by reference into the specification to the same extent asif each individual publication or patent was specifically andindividually indicated to be incorporated herein by reference.

1. An isolated polypeptide monomer of an SK2 calcium-activated potassiumchannel, said monomer forming a potassium channel having a unitconductance of between 2 and 60 pS when a nucleic acid encoding themonomer is expressed in a Xenopus oocyte, wherein said polypeptide isencoded by a nucleic acid that selectively hybridizes under stringentconditions to a sequence of SEQ ID NO:21, wherein the hybridizationreaction is incubated overnight at 37° C. in a solution comprising 40%formamide, 1 M NaCl and 1% SDS, and washed at 55° C. in a solutioncomprising 0.5×SSC.
 2. The polypeptide of claim 1, wherein saidpolypeptide is encoded by a nucleic acid that selectively hybridizesunder stringent conditions to a sequence of SEQ ID NO:15, wherein thehybridization reaction is incubated overnight at 37° C. in a solutioncomprising 40% formamide, 1 M NaCl and 1% SDS, and washed at 55° C. in asolution comprising 0.5×SSC.
 3. The polypeptide of claim 1, comprisingan amino acid sequence selected from the group consisting of SEQ ID NQ:2and SEQ ID NO:19.
 4. The polypeptide of claim 1, wherein saidpolypeptide is encoded by a nucleic acid comprising a nucleotidesequence selected from the group consisting of SEQ ID NO:15 and SEQ IDNO:21.